Baseline Biomonitoring Report

APPENDIX I Wet Season (2018) Baseline Biomonitoring Report

Malingunde ESIA

63 Wessel Road, Rivonia, 2128 PO Box 2597, Rivonia, 2128 South Africa Tel: +27 (0) 11 803 5726 Fax: +27 (0) 11 803 5745 Web: www.gcs-sa.biz

Wet Season Baseline Fish and Mollusc Survey and Biomonitoring Report of the Lilongwe River associated with the Malingunde Flake Graphite Study, Malawi

Report

Version – Final 18 January 2019

Sovereign Metals Limited GCS Project Number: 17 - 1068 Client Reference: GCS Ref: 17 - 1068

GCS (Pty) Ltd. Reg No: 2004/000765/07 Est. 1987 Offices: Durban Gaborone Johannesburg Lusaka Maseru Ostrava Pretoria Windhoek Directors: AC Johnstone (Managing) PF Labuschagne AWC Marais S Napier S Pilane (HR) W Sherriff (Financial) Non-Executive Director: B Wilson-Jones www.gcs-sa.biz Sovereign Metals Limited Aquatic Biomonitoring Assessment

Wet Season Baseline Fish and Mollusc Survey and Biomonitoring Report of the Lilongwe River associated with the Malingunde Flake Graphite Study, Malawi

Report Version – Final

18 January 2019

Sovereign Metals Limited

17-1068

DOCUMENT ISSUE STATUS

Report Issue Final

GCS Reference Number GCS Ref: 17 - 1068

Client Reference GCS Ref: 17 - 1068

Wet Season Baseline Fish and Mollusc Survey and Biomonitoring Report of Title the Lilongwe River associated with the Malingunde Flake Graphite Study, Malawi

Name Signature Date

Author Sandra Carminati 14/01/2019

Document Reviewer Jacques Harris 15/01/2019

Manager sign-off Jacques Harris 15/01/2019

LEGAL NOTICE

This report or any proportion thereof and any associated documentation remain the property of GCS until the mandatory effect’s payment of all fees and disbursements due to GCS in terms of the GCS Conditions of Contract and Project Acceptance Form. Notwithstanding the aforesaid, any reproduction, duplication, copying, adaptation, editing, change, disclosure, publication, distribution, incorporation, modification, lending, transfer, sending, delivering, serving or broadcasting must be authorised in writing by GCS.

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EXECUTIVE SUMMARY

GCS Water and Environmental Consultants (Pty) Ltd (GCS) was requested by Dhamana Consulting, on behalf of Sovereign Metals Limited, to conduct a wet season survey for the proposed Malingunde Flake Graphite Project, 15 km southwest of Lilongwe, Malawi. This work included an aquatic biological survey in the vicinity of the Project area in order to characterise the baseline environmental conditions and support the identification and assessment of environmental and social impacts associated with the proposed Malingunde Project.

The survey included a fish and mollusc baseline survey along with a biomonitoring assessment. The fish and mollusc baseline survey comprised a fish habitat assessment, reference species list compilation and baseline metal analysis. This was undertaken in order to establish the reference species of fish and mollusc currently present in the vicinity of the Project area, to determine baseline conditions with regards to fish habitat availability and to assess the baseline levels of metals occurring within fish. The biomonitoring assessment included a visual survey of the aquatic habitat present at each site, the analysis of in situ water quality, the assessment of general habitat integrity, habitat suitability for the macro- invertebrate community and aquatic macro-invertebrate community integrity, diatom analysis and Whole Effluent Toxicity (WET) testing. This was carried out in order to determine the Present Ecological State (PES) of the aquatic resources in the vicinity of the Project area, to define areas of aquatic ecological sensitivity and to analyse and compare in situ water quality, habitat and community integrity data obtained in April 2017 and February 2018 for the identification and interpretation of any temporal trends in the water quality. Following on from the wet season survey undertaken in April 2017, two dambos, namely the Kankoma Dambo and the Kovuma Dambo, were assessed upstream of the Project area during the wet season survey undertaken in February 2018 to determine baseline conditions within the dambos located in the vicinity of the Project area. Two additional sites, namely Sites MML4 and MML5, were assessed downstream of Site MML3 on the Lilongwe River during the 2018 wet season survey as a result of the expansion of the Project area to the north and east. These sites will serve to indicate any impacts occurring on the Lilongwe River from upstream influences. Four biomonitoring sites were therefore assessed on the Lilongwe River, one upstream of the Project area (the reference site) and three downstream of the Project area to determine any impacts (positive or negative) on this surface water system. One site was assessed on the Lisungwi River, a tributary of the Lilongwe River, to indicate any impact occurring on the Lisungwi River catchment from upstream influences, and in turn provide an indication of any impact on the Lilongwe River from this catchment. A point on a drainage channel within the Project area was also selected to determine baseline conditions within the Project area boundary.

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With regards to the fish and mollusc baseline survey, results of the Fish Habitat Assessment (FHA) conducted during the February 2018 wet season survey indicate that slow-deep conditions dominate at Site MML1, followed by slow-shallow conditions. The fish expected at the site are likely to be limited to fish with a high intolerance for fast-flowing water and shallow habitats. In contrast, fast-shallow, slow-deep and slow-shallow classes occur at Site MML2. The fish expected in this area are likely to be limited to species with a high intolerance for very deep, fast-flowing water. Fast-deep conditions dominate at Site MML3, followed by fast-shallow, slow-deep and slow-shallow conditions in a few small areas. Fish species with a high intolerance for shallow habitats and slow-flowing water are therefore likely to occur at this site. Site MML4 is dominated by slow-deep conditions within the main channel, followed by slow-shallow conditions within some areas along the banks of the system. Fish species with a high intolerance for fast-flowing water and very shallow habitats are likely to occur at this site. Slow-deep conditions dominate at Site MML5 however, the slow-shallow class occurs over a larger area. As a result, the fish community expected to occur at this point are likely to have a high intolerance for very fast-flowing water.

Reference fish species lists for the Lilongwe River, including the Kamuzu Reservoir (as it is on the same system), and the Lisungwi River system, could not be derived from the Fish Reference Frequency of Occurrence (FROC) database (Kleynhans et al., 2007a) or from existing literature as reference information on the frequency of occurrence of fish species within these systems is not available. Therefore, a baseline Ecological Category for fish could not be determined at this time. However, the data obtained during this wet season survey should be utilised as reference values in the Fish Response Assessment Index (FRAI) for Ecological Category determination during future surveys. The reference species list includes 15 indigenous fish species for the Lilongwe River system and 5 indigenous species for the Lisungwi River system. Reference species within the Lilongwe system include Astatotilapia calliptera (Eastern River Bream), Chiloglanis neumanni (Neumann’s Rock Catlet), Clarias gariepinus (Sharptooth Catfish), various Enteromius species including E. choloensis (Silver Barb), E. kerstenii (Redspot Barb), E. macrotaenia (Broadband Barb), E. paludinosus (Straightfin Barb), E. toppini (East Coast Barb) and E. trimaculatus (Threespot Barb); Labeobarbus johnstonii (Johnstonii Yellowfish), Mastacembelus shiranus (Malawi Spinyeel), Opsaridium tweddleorum (Dwarf Sanjika), Oreochromis karongae (Karonga tilapia), Oreochromis lidole (Lidole) and Pseudocrenilabrus philander (Southern Mouthbrooder). Reference species within the Lisungwi River system include Chiloglanis neumanni, Clarias gariepinus, Labeobarbus johnstonii, Mastacembelus shiranus and Oreochromis lidole. Reference species of mollusc within the Lilongwe system include Biomphalaria pfeifferi, Bulinus globosus, Chambardia wahlbergi, Coelatura

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mossambicensis, Gyraulus connollyi, Lanistes ellipticus and Lymnaea natalensis. Bulinus globosus was the only species present within the dambos during the current survey.

Metal analysis conducted on whole fish tissue samples of Astatotilapia calliptera and Oreochromis lidole indicate that the lead (Pb) concentration guideline is exceeded in O. lidole from Site MML1, the chromium (Cr) concentration guideline is exceeded in both A. calliptera and O. lidole from Site MML1 and in O. lidole from Site MML4. This suggests that some impact from existing sources or activities may be occurring at these points during the 2018 wet season survey. With regards to the metal analysis results for the bivalve mollusc, C. mossambicensus, the results indicate that none of the guideline concentration values are exceeded within the mussel tissue. Results of the sediment metal analysis indicate that the chromium (Cr), copper (Cu), iron (Fe) and manganese (Mn) concentration guidelines are exceeded within the sediment sampled from the Kamuzu Reservoir II. This suggests that an impact may be occurring at this point during the current survey.

The in situ water quality at Sites MML1, MML2, MML4 and MML5 during the wet (high flow) season biomonitoring assessment undertaken in February 2018 are considered to be fair. It is evident that the Electrical Conductivity (EC) levels at Sites MML1 and MML2 have increased since the previous wet season survey undertaken in April 2017. As the systems were in flood during the previous survey, it is likely that the nutrients were diluted resulting in the low EC values observed in April 2017. During the survey conducted in February 2018 however, these systems were not in flood, resulting in less dilution and higher readings. Poor water quality conditions are evident at Site MML3, with a significant increase in EC level and an increase in the pH level; and at Sites MMDR, MMD1 KAN and MMD2 KO with an increase in EC levels (significant increases in EC levels have occurred at the two dambo sites) and a decrease in pH and Dissolved Oxygen (DO) levels occurring at these sites in relation to the previous survey. The remainder of the findings of the wet (high flow) season survey conducted in February 2018 are summarised in Table E1 below.

The results of the Intermediate Habitat Integrity Assessment (IHIA) assessment indicate that the general habitat integrity may be regarded as being Moderately Modified (Class C) at Sites MML1, MML2 and MMD1 KAN, Largely Modified (Class D) at Sites MML3 and MML5 and Extensively Modified (Class E) at Sites MML4, MMDR and MMD2 KO. Since the previous wet season survey conducted in April 2017, the general habitat integrity has remained in a Moderately Modified (Class C) condition at Sites MML1 and MML2, has declined from a Largely Natural with Few Modifications (Class B) condition to a Largely Modified (Class D) condition at Site MML3, has declined from an Unmodified, Natural (Class A) condition to a Moderately Modified (Class C) condition at Site MMD1 KAN and from a Largely Modified (Class D) condition to an Extensively Modified (Class E) condition at Site MMD2 KO. The

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habitat integrity at Site MMDR has however improved from a Critically Modified (Class F) condition in April 2017 to an Extensively Modified (Class E) condition during the current survey. The decline in the overall scores obtained at Sites MML1, MML2, MML3, MMD1 KAN and MMD2 KO is mainly a result of increased water quality modifications due to significant increases in EC levels.

The Integrated Habitat Assessment System (IHAS) results indicate that the habitat integrity at Site MML2 is considered adequate in supporting a diverse macro-invertebrate community during the current assessment. The habitat integrity at the remainder of the sites is however inadequate in supporting a diverse community. The habitat integrity at Site MML2 has therefore improved from an inadequate state during the previous survey and has declined from an adequate state at Site MML3. The habitat integrity at the remainder of the sites has remained inadequate since April 2017. The habitat conditions on the Lilongwe River indicate some variation in habitat integrity between the sites, increasing in suitability from MML1 to MML3, after which a decreasing trend is evident further downstream at Sites MML4 and MML5. This is likely a result of the high water levels occurring within the system due to releases from the Kamuzu Reservoir, which is likely to have an influence on the structure of the aquatic communities present at these sites.

The South African Scoring System Version 5 (SASS5) indicates that the macro-invertebrate integrity at Sites MML1, MML4, MML5, MMDR, MMD1 KAN and MMD2 KO is regarded as being in a Severely to Critically Modified (Class E/F) state and in a Largely Modified (Class D) state at Sites MML2 and MML3. Since the previous survey conducted in April 2017, the macro- invertebrate integrity has remained in a Severely to Critically Modified (Class E/F) state at Site MML1, has improved from a Severely to Critically Modified (Class E/F) state to Largely Modified (Class D) state at Site MML2, has declined from a Moderately Modified (Class C) state to a Largely Modified (Class D) state at Site MML3, has remained in a Severely to Critically Modified (Class E/F) state at Site MMDR, and has declined from a Largely Modified (Class D) state to a Severely to Critically Modified (Class E/F) state during the current survey at Sites MMD1 KAN and MMD2 KO.

Results from the diatom analysis indicate that the water quality at each site appeared to have some pollution-related impacts and the overall water quality was Moderate for all sites. According to temporal diatom analysis trends, the ecological water quality has shown a general improvement since April 2017.

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KO, however, was found to pose a slight acute hazard (Class II). Since the previous survey in April 2017, the toxicological hazard has improved from a slight acute hazard (Class II) condition to a no acute hazard (Class I) condition at Site MML1, from an acute hazard (Class III) condition to a no acute hazard (Class I) condition at Sites MML2 and MML3 and from a high acute hazard (Class IV) condition to a no acute hazard (Class I) condition at Site MMDR during the current assessment. The toxicological hazard has remained in a slight acute hazard (Class II) condition at Sites MMD1 KAN and MMD2 KO.

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Table E1: Summary of the Results for the Wet (High Flow) Season Survey in February 2018

Survey MML1 MML2 MML3 MML4 MML5 MMDR MMD1 KAN MMD2 KO

IHIA Class C C D E D E C E

IHAS Class Inadequate Adequate Inadequate Inadequate Inadequate Inadequate Inadequate Inadequate

SASS5 Class E/F D D E/F E/F E/F E/F E/F

ASPT Score 4.6 5.9 5.8 4.1 3.8 3.4 4.1 3.8

Diatom Analysis Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate

No Acute No Acute No Acute No Acute No Acute No Acute Slight Acute Slight Acute WET Hazard Hazard Hazard Hazard Hazard Hazard Hazard Hazard Hazard Classification Class I Class I Class I Class I Class I Class I Class II Class II

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The following is recommended as part of the current wet (high flow) season fish and mollusc baseline survey and aquatic biomonitoring assessment:

It is recommended that a bi-annual biomonitoring program i.e. a wet season and dry season survey, be implemented every year going forward in order to closely monitor any impacts resulting from the proposed mining activities over time. This will enable the implementation of effective control measures in order to manage and control any impacts that may compound upon the existing impacts already occurring on the water resources in the vicinity of the Project area. A wet season survey should be implemented in February 2019 to remain consistent with this survey and for data to be compared temporally to the results of this document in order to identify any trends in the water quality. The survey should also include an assessment of the fish community integrity in order to utilise the reference fish data obtained during the current survey to determine the baseline Ecological Category for fish within the river reach and monitor the Ecological Category going forward;

The monitoring of spatial and temporal variations in salt loads and pH levels should be carried out at all sites. Considering the findings obtained from the baseline metal analysis during the current assessment, the metal concentrations within fish tissue, bivalve tissue (if possible, depending on availability) and sediment should also be monitored at all sites in order to provide a comprehensive representation of the levels occurring within the aquatic ecosystems in the vicinity of the Project area; and

Definitive toxicological testing of the process water associated with the proposed Malingunde Flake Graphite Project should be carried out on an annual basis in order to determine the rate at which potential discharges should take place without severely negatively affecting the receiving aquatic environment.

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CONTENTS PAGE

1. INTRODUCTION ...... 16 2. SCOPE OF WORK ...... 18 3. METHODOLOGY ...... 21 3.1. ENVIRONMENTAL CONTEXT OF THE PROJECT AREA ...... 21 3.2. VISUAL SURVEY ...... 21 3.3. IN SITU WATER QUALITY ...... 21 3.3.1. pH...... 22 3.3.2. Temperature ...... 22 3.3.3. Electrical Conductivity (EC) and Total Dissolved Solids (TDS)...... 23 3.3.4. Dissolved Oxygen (DO) ...... 23 3.4. HABITAT INTEGRITY ...... 24 3.4.1. General Habitat Integrity ...... 24 3.4.2. Habitat Suitability ...... 24 3.5. AQUATIC MACRO-INVERTEBRATE COMMUNITY INTEGRITY ASSESSMENT ...... 25 3.6. FISH COMMUNITY INTEGRITY ASSESSMENT ...... 27 3.6.1. Fish Habitat Assessment (FHA) ...... 27 3.6.2. The Fish Response Assessment Index (FRAI) ...... 28 3.7. MOLLUSC ASSESSMENT ...... 31 3.8. METAL ANALYSIS ...... 31 3.9. DIATOM ANALYSIS ...... 33 3.10. WHOLE EFFLUENT TOXICITY (WET) TESTING ...... 34 3.10.1. Sample Preparation ...... 35 3.10.2. Detailed Methodologies ...... 35 3.10.3. Toxicity Units...... 39 4. RESULTS & DISCUSSION ...... 40 4.1.1. Environmental Context of the Project Area ...... 40 4.2. VISUAL SURVEY ...... 45 4.3. IN SITU WATER QUALITY RESULTS ...... 54 4.4. HABITAT INTEGRITY ...... 59 4.4.1. General Habitat Integrity ...... 59 4.4.2. Habitat Suitability ...... 63 4.5. AQUATIC MACRO-INVERTEBRATE COMMUNITY INTEGRITY ASSESSMENT ...... 66 4.6. FISH COMMUNITY INTEGRITY ASSESSMENT ...... 76 4.6.1. Fish Habitat Assessment (FHA) ...... 76 4.6.2. The Fish Response Assessment Index (FRAI) ...... 78 4.6.2.1. Reference Conditions ...... 78 4.7. MOLLUSC ASSESSMENT ...... 80 4.8. METAL ANALYSIS ...... 81 4.9. DIATOM ANALYSIS ...... 85 4.9.1. Site MML1 ...... 85 4.9.2. Site MML2 ...... 86 4.9.3. Site MML3 ...... 86 4.9.4. Site MML4 ...... 87 4.9.5. Site MML5 ...... 87 4.9.6. Site MMDR ...... 88 4.9.7. Site MMD1 KAN ...... 88 4.9.8. Site MMD2 KO ...... 88 4.10. WHOLE EFFLUENT TOXICITY (WET) TESTING ...... 94 5. CONCLUSION ...... 97 6. RECOMMENDATIONS ...... 102

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7. REFERENCES ...... 103 8. APPENDICES ...... 112

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List of Figures

Figure 2-1: Aquatic Biomonitoring Monitoring Points for the Malingunde Flake Graphite Project ...... 20 Figure 3-1: Ecological Categories according to the EcoStatus A to F Continuum Approach ...... 30 Figure 4-1: Water Resource and Major Catchment Areas in the vicinity of the Project Area ...... 42 Figure 4-2: Major Wetlands and Reserves in the vicinity of the Project Area ...... 44 Figure 4-3: Upstream view in a southerly direction indicating the high flow of the river at this point and the good cover provided by aquatic macrophytes...... 46 Figure 4-4: Downstream view in a northerly direction indicating the slow flowing system at the time of assessment...... 46 Figure 4-5: Upstream view in a south-easterly direction indicating fast runs occurring in the vicinity of the bridge and flowing downstream. Overhanging marginal vegetation in the form of reed stems is also evident...... 47 Figure 4-6: Downstream view in a north-westerly direction indicating the excellent stones biotope observed within a rapid which culminates in deep, slow-flowing pools beyond this point...... 47 Figure 4-7: Upstream view in a south-westerly direction indicating the presence of deep pool habitat and the pillars of the road bridge...... 48 Figure 4-8: Downstream view in a north-easterly direction indicating the fast flow of the system and excellent bank cover present at this site...... 48 Figure 4-9: Upstream view in a southerly direction indicating the high water levels observed within the channel during the survey...... 49 Figure 4-10: Sand harvesting and trench formation evident on the left bank of the channel...... 49 Figure 4-11: Downstream view in a north-easterly direction indicating the excellent bank cover provided by the marginal vegetation at this site...... 49 Figure 4-12: Sand harvesting and extensive erosion of the right bank...... 49 Figure 4-13: Upstream view in a south-westerly direction indicating the deep, slow-flowing run and excellent bank cover at this site...... 50 Figure 4-14: Downstream view in a north-westerly direction indicating the incision and erosion of the left bank of the channel...... 50 Figure 4-15: Upstream view in a northerly direction indicating the low water levels occurring within the drainage line at the time of the assessment...... 51 Figure 4-16: Downstream view in a southerly direction indicating the stagnant water within the stream...... 51 Figure 4-17: North-easterly view of the Kankoma dambo indicating the small, shallow pool observed during the survey...... 52 Figure 4-18: North-easterly view of the Kankoma dambo indicating the overgrazing of indigenous vegetation by livestock resulting in soil erosion...... 52 Figure 4-19: North-easterly view of the Kovuma dambo indicating the small, stagnant pool of silty water containing aquatic macrophytes...... 53 Figure 4-20: North-easterly view of the Kovuma dambo indicating the presence of marginal vegetation alongside the road bridge that crosses the site...... 53 Figure 4-21: Spatial Variation in Water Quality between the Monitoring Points on the Aquatic Resources in the vicinity of the Project Area in February 2018...... 56 Figure 4-22: Temporal Variation in Electrical Conductivity between the Wet Season Surveys undertaken in April 2017 and February 2018 at the Biomonitoring Sites ...... 57 Figure 4-23: Temporal Variation in pH between the Wet Season Surveys undertaken in April 2017 and February 2018 at the Biomonitoring Sites ...... 58 Figure 4-24: Temporal Variation in Dissolved Oxygen between the Wet Season Surveys undertaken in April 2017 and February 2018 at the Biomonitoring Sites ...... 58 Figure 4-25: Temporal Variation in Temperature between the Wet Season Surveys undertaken in April 2017 and February 2018 at the Biomonitoring Sites ...... 59 Figure 4-26: Spatial Variation in SASS5, IHAS and ASPT Scores between the Monitoring Points on the Aquatic Resources in the vicinity of the Project Area in February 2018 ...... 69

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Figure 4-27: Temporal Variation in SASS5, IHAS and ASPT Scores obtained during the Wet Season Surveys undertaken in April 2017 and February 2018 at Site MML1. The Variation in EC and pH Levels are also indicated for ease of reference ...... 71 Figure 4-28: Temporal Variation in SASS5, IHAS and ASPT Scores obtained during the Wet Season Surveys undertaken in April 2017 and February 2018 at Site MML2. The Variation in EC and pH Levels are also indicated for ease of reference ...... 72 Figure 4-29: Temporal Variation in SASS5, IHAS and ASPT Scores obtained during the Wet Season Surveys undertaken in April 2017 and February 2018 at Site MML3. The Variation in EC and pH Levels are also indicated for ease of reference ...... 73 Figure 4-30: Temporal Variation in SASS5, IHAS and ASPT Scores obtained during the Wet Season Surveys undertaken in April 2017 and February 2018 at Site MMDR. The Variation in EC and pH Levels are also indicated for ease of reference ...... 74 Figure 4-31: Temporal Variation in SASS5, IHAS and ASPT Scores obtained during the Wet Season Surveys undertaken in April 2017 and February 2018 at Site MMD1 KAN. The Variation in EC and pH Levels are also indicated for ease of reference ...... 75 Figure 4-32: Temporal Variation in SASS5, IHAS and ASPT Scores obtained during the Wet Season Surveys undertaken in April 2017 and February 2018 at Site MMD2 KO. The Variation in EC and pH Levels are also indicated for ease of reference ...... 75 Figure 4-33: The Quantity and Diversity of Cover Available for the Fish Community at each Site for each Velocity-Depth Class during the Wet (High Flow) Season in February 2018 ...... 76 Figure 4-34: Temporal Variation in %PTV for the Monitoring Points on the Aquatic Resources in the vicinity of the Project Area from April 2017 to February 2018 ...... 93

List of Tables

Table 2-1: Location of the Aquatic Sampling Sites ...... 19 Table 3-1: Classification of Present Ecological State Classes in terms of General Habitat Integrity (Kemper, 1999) ...... 24 Table 3-2: Reference Conditions for Sandy and Rocky River Systems based ASPT scores (Tambala et al., 2016) ...... 27 Table 3-3: Velocity-Depth and Cover Classes ...... 27 Table 3-4: Abundance Scoring of Velocity-Depth and Cover Classes (Adapted from Rankin, 1995)...... 28 Table 3-5: Classification of Ecological Categories in line with the River Health Program ...... 30 Table 3-6: Limit and Class Values used for Diatom Indices in the Evaluation of Water Quality [Adapted from Eloranta & Soininen (2002)]...... 34 Table 3-7: Interpretation of the % PTV Scores (Adapted from Kelly, 1998) ...... 34 Table 3-8: Summary of the Test Conditions and Test Acceptability Criteria for the Vibrio fischeri and Selenastrum capricornutum Acute Toxicity Screening Tests (Biotox, 2018) ...... 37 Table 3-9: Summary of the Test Conditions and Test Acceptability Criteria for the Daphnia magna and Poecilia reticulata Acute Toxicity Screening Tests (Biotox, 2018) ...... 38 Table 3-10: Hazard Classification System for Daphnia magna and Poecilia reticulata Screening Tests . 39 Table 3-11: Grouping of Toxicity Units ...... 39 Table 4-1: Summary of Descriptions and Photographs of Each Site for the High Flow Survey in February 2018 ...... 46 Table 4-2: In situ Water Quality Results for the Wet Season Survey in February 2018 ...... 54 Table 4-3: IHIA Results for the Wet (High Flow) Season in February 2018 ...... 60 Table 4-4: IHIA Results for the Wet (High Flow) Season in April 2017 ...... 60 Table 4-5: IHAS Results for the Wet (High Flow) Season in February 2018 ...... 63 Table 4-6: IHAS Results for the Wet (High Flow) Season in April 2017 ...... 65 Table 4-7: SASS5 Results for the Wet (High Flow) Season in February 2018 ...... 67 Table 4-8: SASS5 Results for the Wet (High Flow) Season in April 2017 ...... 70 Table 4-9: Reference Fish Species List derived during the February 2018 Wet (High Flow) Season for the Lilongwe and Lisungwi Rivers ...... 79

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Table 4-10: Reference Mollusc Species List derived during the February 2018 Wet (High flow) Season for the Lilongwe River Reach and the Two Dambo Systems ...... 81 Table 4-11: Baseline Metal Analysis Results of Whole Fish Tissue Sampled from the Lilongwe River, the Lisungwi River and the Kamuzu Reservoir II during the Wet (High Flow) Season in February 2018 82 Table 4-12: Baseline Metal Analysis Results of the Native Bivalve Mollusc, Coelatura mossambicensus, sampled from Site MML4 on the Lilongwe River during the Wet (High Flow) Season in February 201883 Table 4-13: Metal Analysis Results in Sediment Sampled from the Kamuzu Reservoir II during the Wet (High Flow) Season in February 2018 ...... 84 Table 4-14: Species List for each Site indicating Abundance and Dominance of Diatom Species in February 2018 ...... 90 Table 4-15: Diatom Index Scores for the Study Sites indicating the Ecological Water Quality obtained in February 2018 ...... 92 Table 4-16: Diatom Index Scores for the Study Sites indicating the Ecological Water Quality in October 2017 (Walsh, November 2017) ...... 93 Table 4-17: Diatom Index Scores for the Study Sites indicating the Ecological Water Quality in April 2017 (Walsh, June 2017) ...... 93 Table 4-18: Diatom Index Scores for the Study Sites indicating the Ecological Water Quality Trends between Seasons ...... 94 Table 4-19: Summary of the Results obtained for Toxicological Testing in February 2018 ...... 95

List of Appendices

APPENDIX A: METAL ANALYSIS REPORT (FEBRUARY 2018) ...... 112 APPENDIX B: DIATOM ANALYSIS REPORT (FEBRUARY 2018) ...... 123 APPENDIX C: TOXICOLOGICAL REPORT (FEBRUARY 2018) ...... 145

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GLOSSARY AND ABREVIATIONS

Aquatic Ecology ASPT Average Score Per Taxa BGIS Biodiversity Geographic Information Systems BDI Biological Diatom Index DO Dissolved Oxygen EC Electrical Conductivity EPT Index Ephemeroptera, Plecoptera and Trichoptera Index FHA Fish Habitat Assessment FHR Fish Habitat Rating FRAI Fish Response Assessment Index FROC Fish Frequency of Occurrence GSM Gravel, Sand and Mud Heterogeneous Diverse in character or content. IHAS Integrated Habitat Assessment System IHIA Intermediate Habitat Integrity Assessment MBS Malawi Bureau of Standards Phyla A biological taxon between Kingdom and Class PES Present Ecological State PTV Pollution Tolerant Valves SASS5 South African Scoring System Version 5 SPI Specific Pollution Index Taxa A taxonomic category or group TDS Total Dissolved Solids US EPA United States Environmental Protection Agency WET Whole Effluent Toxicity WHO World Health Organization Ecotoxicology Bioluminescent The production and emission of light by a living organism Daphnia magna Common species of water flea representing the invertebrate trophic level DEEEP Direct Estimation of Ecological Effect Potential LC10 Lethal Concentration at which 10% of the test population will perish LC50 Lethal Concentration at which 50% of the test population will perish Luminescence The emission of light by a substance that has not been heated Lyophilised Freeze-drying Neonate New born

Poecilia reticulata A small, colourful tropical species of freshwater fish (Guppy) Selenastrum capricornutum A species of green microalgae NaCl Sodium Chloride. Also known as salt or halite Toxicity The degree to which a substance can harm an organism Vibrio fischeri A species of bioluminescent bacterium

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1. INTRODUCTION

The aquatic biological wet season survey was conducted from the 12th to the 19th of February 2018 and included a fish and mollusc baseline survey along with a biomonitoring assessment. Following on from the wet season survey undertaken in April 2017, two dambos, namely the Kankoma Dambo and the Kovuma Dambo, were assessed upstream of the Project area during the wet season survey undertaken in February 2018 to determine baseline conditions within the dambos located in the vicinity of the Project area. Two additional sites, namely Sites MML4 and MML5, were assessed downstream of Site MML3 on the Lilongwe River during the 2018 wet season survey as a result of the expansion of the Project area to the north and east. These sites will serve to indicate any impacts occurring on the Lilongwe River from upstream influences. Four biomonitoring sites were therefore assessed on the Lilongwe River, one upstream of the Project area (the reference site) and three downstream of the Project area to determine any impacts (positive or negative) on this surface water system. One site was assessed on the Lisungwi River, a tributary of the Lilongwe River, to indicate any impact occurring on the Lisungwi River catchment from upstream influences, and in turn provide an indication of any impact on the Lilongwe River from this catchment. A point on a drainage channel within the Project area was also selected to determine baseline conditions within the Project area boundary.

Biological indicators provide a means of determining the effects of changes in water quality on whole ecosystems. This involves utilising living organisms as indicators of disturbance in an ecosystem and this method has been proven successful (Rosenberg and Resh, 1993). In fact, bio-assessment has been acclaimed as a more sensitive and reliable measure of environmental conditions than physical or chemical measurements (Warren, 1971).

The fish and mollusc baseline survey comprised a fish habitat assessment, reference species list compilation and baseline metal analysis. This was undertaken in order to establish the reference species of fish and mollusc currently present in the vicinity of the Project area, to determine baseline conditions with regards to fish habitat availability and

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to assess the baseline levels of metals occurring within fish. The biomonitoring assessment included a visual survey of the aquatic habitat present at each site, the analysis of in situ water quality, the assessment of general habitat integrity, habitat suitability for the macro-invertebrate community and aquatic macro-invertebrate community integrity, diatom analysis and Whole Effluent Toxicity (WET) testing. This was carried out in order to determine the Present Ecological State (PES) of the aquatic resources in the vicinity of the Project area, to define areas of aquatic ecological sensitivity and to analyse and compare in situ water quality, habitat and community integrity data obtained in April 2017 and February 2018 for the identification and interpretation of any temporal trends in the water quality. It must be noted that the assessment of the macro-invertebrate integrity using the Ephemeroptera, Plecoptera and Trichoptera (EPT) index was not carried out in this study as this protocol strongly relies on the presence of Ephemeroptera, Plecoptera and Trichoptera, which were not observed at all of the sites i.e. they were absent at Sites MML1, MML4, MML5, MMDR, MMD1 KAN, and MMD2 KO. Therefore, the South African Scoring System Version 5 (SASS5) protocol was regarded as more applicable and utilised in this assessment.

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2. SCOPE OF WORK

The purpose of the aquatic assessment was to characterise the baseline aquatic environmental conditions and support the identification and assessment of environmental and social impacts associated with the proposed Malingunde Project by determining the PES of the aquatic resources associated with the Project area.

The objectives of the survey were to:

• Determine the PES of the surface water system;

• Highlight any significant trends in the water quality;

• Define areas of aquatic ecological sensitivity;

• Analyse and compare data obtained during the biomonitoring assessments conducted in April 2017 and February 2018 for the identification of any temporal trends in the water quality;

• Determine baseline conditions with regards to fish and mollusc species present in the vicinity of the Project area; and

• Provide recommendations for future monitoring programs.

The biomonitoring sites are located on the Lilongwe River, the Lisungwi River, a drainage channel within the Project area and within two dambos in proximity to the Project. One site, Site MML1, was assessed upstream of the Project area on the Lilongwe River. Two sites, Sites MMD1 KAN and MMD2 KO, were assessed upstream of the Project area to the north of the watershed. One site, Site MML2, was assessed upstream of the Project area on the Lisungwi River, a tributary of the Lilongwe River. Three sites, namely Sites MML3, MML4 and MML5 were assessed downstream of the Project area on the Lilongwe River. One site, Site MMDR was also assessed on a drainage channel within the Project area. Eight toxicity sample points were also selected to correspond with each biomonitoring site and were analysed on four trophic levels. The diatom assemblages were also analysed at each site in order to detect specific changes in environmental conditions and provide an overall indication of trends within the aquatic systems (Walsh, 2017). Baseline metal analysis was conducted within fish, bivalve and sediment where possible, in order to determine baseline concentrations of metals within the aquatic ecosystems in the vicinity of the Project area.

The positions of the biomonitoring and toxicity sampling points in relation to the proposed Malingunde Project area are presented in Figure 2-1. Table 2-1 provides geographical location information, along with a description of each point.

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Table 2-1: Location of the Aquatic Sampling Sites

UTM 36 S Site Description X Y Situated on the upper section of the Lilongwe River, upstream of the Project area and the Kamuzu 566381.419 8427953 MML1 Reservoir. This site serves to indicate the state of the

system prior to any influence from the Project area and reservoir.

Located in the lower section of the Lisungwi River, a tributary of the Lilongwe River. This site serves to indicate any impact occurring on the Lisungwi River 574476.692 8434100 MML2 from upstream sources unrelated to the Project area, which may in turn impact on the Lilongwe River catchment.

Located on the Lilongwe River, downstream of Sites MML1, MML2 and the confluence with the Lisungwi 574689.708 8435476 MML3 River. An indication of the state of the system following

any influence from the Project area, the Kamuzu Reservoir and the Lisungwi River is provided at this site.

Located on the Lilongwe River, downstream of Sites MML1, MML2 and MML3. This site serves to indicate any MML4 576126.75 8438244 impact occurring on the Lilongwe River from upstream sources including the Project area.

Located on the Lilongwe River, downstream of Sites MML1, MML2, MML3 and MML4. This site provides an MML5 576338.35 8439431 indication of any cumulative impacts from upstream sources including the Project area.

Located on a drainage channel within the Project area boundary. An indication of the state of the system 571810.371 8435452 MMDR within the Project area boundary is provided at this site.

Located approximately 1.3 km upstream of the Project area on the opposite side of the watershed. This site MMD1 570573.266 8440136 KAN serves to indicate the state of the dambos prior to any influence from the Project area.

Located approximately 2 km upstream of the Project area on the opposite side of the watershed. This site MMD2 572041.35 8438449 KO serves to indicate the state of the dambos prior to any influence from the Project area.

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Figure 2-1: Aquatic Biomonitoring Monitoring Points for the Malingunde Flake Graphite Project

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3. METHODOLOGY

The following methodology was adhered to in the aquatic biomonitoring assessment.

3.1. Environmental Context of the Project Area

A detailed desktop survey of the information available for the Project area was conducted in order to obtain an overview of the environmental context of the area. This included information on climate, topography, relief, slope, water resource management, ecological importance, sensitivity and conservation value with regards to freshwater ecosystems within, or in close proximity to, the Project area. This was undertaken by reviewing the data available for the specific region within which the Project area occurs.

3.2. Visual Survey

A visual survey of each biomonitoring site was carried out during the assessment. Both instream and riparian zone factors were recorded. Instream characteristics included the type of biotopes present, diversity in flow velocity and depth of flow, visual attributes of the water, and types of macrophytes and fauna present. Riparian zone characteristics recorded included the type of riparian vegetation present, stream bank incision and potential for erosion and the presence of man-made structures such as gabions, weirs and bridges. Photographs were taken at each site to document the status of the site during the assessment.

Visible impacts from surrounding anthropogenic activities and any natural limitations on the aquatic system were also noted. In general, examples of such natural limitations include a lack of habitat diversity due to the dominance of bedrock at the site and a lack of flow due to the non-perennial nature of the system.

3.3. In Situ Water Quality

According to Palmer et al. (2004), water quality may be defined as the combined effects of the physical attributes and the chemical constituents of an aquatic ecosystem. Water quality has a direct impact on the aquatic biota residing within river systems and therefore the data obtained during in situ testing is used to aid in the interpretation of the biomonitoring results. The biota-specific water quality variables measured during the assessment include pH, temperature, Dissolved Oxygen (DO) and Electrical Conductivity (EC). The in situ water quality results were compared against the guidelines specified by the Malawi Bureau of Standards (MBS) (2005), the World Health Organization (WHO) (2011)

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and the Target Water Quality Ranges (TWQRs) in terms of The South African Water Quality Guidelines (SAWQG) for Aquatic Ecosystems, Volume 7 (DWAF, 1996). The South African guidelines provide specific standards for spatial and temporal variations in the water quality variables. The in situ field measurements are discussed below.

3.3.1. pH

The pH of natural waters is determined by both geological and atmospheric influences, as well as by biological activities. Most fresh waters are usually relatively well buffered with a pH range from 6 to 8 (Davies and Day, 1998) and are slightly alkaline due to the presence of bicarbonates of the alkali and alkaline earth metals (DWAF, 1996). The pH target for fish health should range between 6.5 and 9.0, as most species will tolerate and reproduce successfully within this pH range (Alabaster and Lloyd, 1982). A pH value of > 9.0 usually indicates eutrophic conditions (nutrient enrichment) (Davies and Day, 1998). The nutrient loads that cause eutrophication are usually a consequence of human activities and may result from runoff from farms, industrial, urban and animal waste. According to the MBS (2005), the acceptable pH range for drinking water is between 5 and 9.5. However, this range is not considered as an acceptable guideline for aquatic ecosystems due to the reasons mentioned above. According to the WHO (2011), the acceptable pH range is between 6.5 and 8.5, which falls within the acceptable range for aquatic ecosystems. This range was therefore considered an appropriate guideline range and adhered to for the Project. In addition, spatial and temporal variations in pH level along a watercourse should not vary by more than 5% (DWAF, 1996), as this may limit the integrity of the aquatic community.

3.3.2. Temperature

Water temperature plays an important role in aquatic ecosystems by affecting the rates of chemical reactions and therefore also the metabolic rates of organisms (Davies and Day, 1998). Temperature affects the overall physiological processes of organisms, such as the rate of development, reproductive periods and emergence time of organisms (Davies and Day, 1998). Temperature varies with season and the life cycles of many aquatic macroinvertebrates are cued by temperature (Davies and Day, 1998).

Aquatic organisms have upper and lower thermal tolerance limits, an optimal temperature for growth, a preferred temperature range in thermal gradients, and temperature limitations for migration, spawning and egg incubation. Therefore, rapid changes in temperature may severely affect aquatic organisms and lead to mass mortality. Less severe temperature changes in water bodies may have sub-lethal effects or lead to an alteration in the existing aquatic community. The temperatures of inland waters in South Africa

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generally range from 5 to 30 C̊ (DWAF, 1996). As no reference information is available on the temperature ranges of inland waters in Malawi, and standard guidelines for temperature are not specified according to the MBS (2005) and the WHO (2011), the above target range of 5 to 30 C̊ was considered an appropriate guideline range and adhered to for the Project.

3.3.3. Electrical Conductivity (EC) and Total Dissolved Solids (TDS)

Electrical conductivity (EC) is a measure of the ability of water to conduct an electrical current (DWAF, 1996). This ability is a result of the presence in water of ions such as carbonate, bicarbonate, chloride, sulphate, nitrate, sodium, potassium, calcium and magnesium, all of which carry an electrical charge (DWAF, 1996). Many organic compounds that dissolve in water do not dissociate into ions (ionise), and consequently they do not affect the EC (DWAF, 1996). EC is a rapid and useful surrogate measure of the Total Dissolved Solids (TDS) concentration of waters with a low organic content (DWAF, 1996). For the purpose of interpretation of the biological results collected during the survey, the TDS concentrations were calculated using the following generic constant (DWAF, 1996):

TDS (mg/l) = EC (mS/m at 25 ˚C) x 6.5

Where: mg/l = milligrams per litre; and

mS/m = millisiemens per metre.

If more accurate estimates of the TDS concentration from EC measurements are required then the conversion factor should be experimentally determined for each specific site and for specific runoff events (DWAF, 1996). According to Davies and Day (1998), freshwater organisms usually occur where TDS values are less than 3000 mg/l. According to the MBS (2005) and the WHO (2011), TDS levels of less than 1000 mg/l are considered acceptable. According to the MBS (2005), EC levels of less than 150 mS/m are considered acceptable. In addition, spatial and temporal variations in EC level along a watercourse should not vary by more than 15%, as this may lead to osmotic stress within aquatic communities (DWAF, 1996).

3.3.4. Dissolved Oxygen (DO)

The maintenance of adequate Dissolved Oxygen (DO) is critical for the survival and functioning of aquatic biota as it is required for the respiration of all aerobic organisms. Therefore, the DO concentration provides a useful measure of the health of an ecosystem (DWAF, 1996). As no guidelines for DO are specified according to the MBS (2005) and the

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WHO (2011), the median guideline for DO of more than 5 mg/l for the protection of aquatic biota was utilised (Kempster et al., 1980).

3.4. Habitat Integrity

3.4.1. General Habitat Integrity

The general habitat integrity of each site was assessed using the Intermediate Habitat Integrity Assessment (IHIA) developed by Kleynhans (1996) and adapted by Kemper (1999) for application in rapid intermediate habitat assessments. Results obtained from this index were used to aid in the interpretation of the biotic integrity results. The method assesses the PES of both the instream and riparian zone habitat integrity in terms of impacts such as water abstraction, flow and channel modifications, inundation and water quality. Scores are allocated according to the extent of the impact related to each factor and total scores for instream and riparian zone integrity are summed and averaged to provide an overall percentage for the PES of the general habitat integrity. The method classifies the PES into one of six classes, ranging from Unmodified/Natural (Class A), to Critically Modified (Class F) (Table 3-1).

Table 3-1: Classification of Present Ecological State Classes in terms of General Habitat Integrity (Kemper, 1999)

Class Description Score (% of total)

A Unmodified, Natural. 90-100

B Largely Natural, with Few Modifications. 80-89

C Moderately Modified. 60-79

D Largely Modified. 40-59

E Extensively Modified. 20-39

F Critically Modified. <20

3.4.2. Habitat Suitability

The Integrated Habitat Assessment System Version 2 (IHAS v2) was developed by McMillan (1998) for use in conjunction with the SASS5 protocol. The IHAS was applied at each biomonitoring site in order to assess the specific habitat suitability for aquatic macro- invertebrates and aid in the interpretation of the SASS5 results. The IHAS scoring system is divided into two sections, namely the sampling habitat, comprising 55% of the total score,

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and the general stream condition, comprising 45% of the total score. Summation of the scores obtained for the two sections provide an overall habitat percentage. Scores for the IHAS index were interpreted according to the following guidelines:

• <65%: habitat diversity and structure is inadequate for supporting a diverse aquatic macro-invertebrate community;

• 65% to 75%: habitat diversity and structure is adequate for supporting a diverse aquatic macro-invertebrate community; and

• >75%: habitat diversity and structure is highly suited for supporting a diverse aquatic macro-invertebrate community.

3.5. Aquatic Macro-Invertebrate Community Integrity Assessment

The monitoring of the integrity of the macro-invertebrate community of an aquatic ecosystem forms an integral part in monitoring of integrity of that ecosystem for the following reasons:

The relatively sedentary nature of the community that enables the detection of localised disturbances; The relatively long life-cycles of ±1 year that allows for the integration of pollution effects over time; The ease with which field sampling is carried out; and The heterogeneity of the community allows for several phyla to be represented, and therefore responses to environmental impacts are detectable in terms of the community as a whole (Hellawell, 1977).

The SASS5 index was designed specifically for the evaluation of low/moderate flow hydrology and is not applicable in wetlands, impoundments, estuaries and other lentic habitats (Dickens and Graham, 2002). The standard SASS5 sampling methodology was applied as defined by Dickens and Graham (2002) by an accredited River Eco-Status Monitoring Programme (REMP) practitioner.

The endpoint of any biological or ecosystem assessment is a value expressed, either in the form of measurement i.e. data collected, or by summarising the measurements into one or several indices. The endpoint values used in this study are the total SASS5 score, providing an indication of the diversity of the macro-invertebrate community, and the Average Score Per Taxon (ASPT), indicating community sensitivity. As the purpose of this study is to

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characterise the current ecological status of the aquatic ecosystem, the current survey results were compared to those of previous surveys.

According to Dickens and Graham (2002), the index is based on the presence of aquatic invertebrate families and the perceived sensitivity to water quality changes of these families. Different families show different sensitivities to pollution. These sensitivities range from highly tolerant families such as the Family Muscidae, specifically House flies and Family Psychodidae, specifically Moth flies; to highly sensitive families such as the Family Oligoneuridae, specifically Brushlegged mayflies.

According to Van der Merwe (2003), a broad explanation of the sensitivity scales are as follows:

1 to 5: Highly tolerant to pollution, e.g. Family Baetidae, score: 4; 6 to 10: Moderately tolerant to pollution, e.g. Family Ecnomidae, score: 8; and 11 to 15: Very low tolerances to pollution, e.g. Family Heptageniidae, score: 13.

Results from the IHAS Index were used to aid in the interpretation of the SASS5 results by considering the effects of habitat variation on the macro-invertebrate community integrity. This is because the three biotopes incorporated in the SASS5 protocol, namely, stones and bedrock; gravel, sand and mud; and marginal and aquatic riparian vegetation in and out of current may not be present in all river systems. Therefore, a system with a low SASS5 score along with a low IHAS score may not necessarily reflect poor water quality. Rather, the low SASS5 score may be attributed to a lack of habitat diversity. Furthermore, the water quality in a system that has achieved a high SASS5 score with a high IHAS score may not be as pristine as a system achieving a high SASS5 score with a low IHAS score. In this case, the diversity of the macro-invertebrate community is high despite a low IHAS score, likely indicating good water quality. However, a system that achieves a high IHAS score along with a low SASS5 score is likely to reflect poor water quality. This is because the habitat is not a limiting factor and the low SASS5 score is therefore likely to be attributed to the poor water quality of the system. Confirmation of this result may be made by taking the ASPT score into consideration as it provides an indication of the sensitivity of the macro- invertebrate community.

Table 3-2 presents the interpretation guidelines used for this assessment which is based on the methodology applied by Tambala et al. (2016) in order to retain consistency for comparative purposes. Based on the methodology described, ASPT scores of more than 6.9 attained in sandy rivers and more than 7.9 attained in rocky rivers are regarded as the reference scores i.e. reflect reference conditions. The current results were therefore interpreted in relation to these reference scores.

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Table 3-2: Reference Conditions for Sandy and Rocky River Systems based ASPT scores (Tambala et al., 2016)

Class Description ASPT ASPT (Sandy) (Rocky) A Unmodified, Natural. >6.9 >7.9

B Largely Natural, with Few Modifications. 5.8 – 6.9 6.8 – 7.9

C Moderately Modified. 4.9 – 5.8 6.1 – 6.8

D Largely Modified. 4.3 – 4.9 5.1 – 6.1

E/F Seriously to Critically Modified. <4.3 <5.1

3.6. Fish Community Integrity Assessment

3.6.1. Fish Habitat Assessment (FHA)

The Fish Habitat Assessment (FHA) evaluates the potential that the habitat at a given site possesses to provide suitable conditions for a fish species to occur there. This provides a framework within which the presence, absence and frequency of occurrence of species can be interpreted. The FHA was carried out by assessing the habitat according to the diversity of the velocity-depth classes and the presence of various types of cover within each of these velocity-depth classes. Any impacts that may influence the habitat integrity for fish were also considered (Kleynhans, 2007). The four velocity-depth classes assessed at each site are provided in Table 3-3, along with five cover classes.

Table 3-3: Velocity-Depth and Cover Classes

Velocity-Depth Classes Cover Classes

Slow-Deep Overhanging Vegetation

Slow-Shallow Undercut Banks and Root Wads

Fast-Deep Stream Substrate

Aquatic Macrophytes Fast-Shallow Water Column

Notes: Slow: <0.3 m/s; Fast: >0.3 m/s; Shallow: <0.5 m; Deep: >0.5 m.

The relative abundance of these classes was rated at each site according to the scores provided in Table 3-4.

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Table 3-4: Abundance Scoring of Velocity-Depth and Cover Classes (Adapted from Rankin, 1995)

Occurrence Descriptor Relative Abundance Score (% of Area Covered)

None 0 0

Rare 0-5 1

Sparse 5-25 2

Common 25-75 3

Abundant 75-90 4

Very Abundant 90-100 5

The Fish Habitat Rating (FHR) was determined for each site by calculating the contribution of each velocity-depth class at the site and multiplying this value by the sum of each cover class available for each velocity-depth class:

FHR = vd/vd x c

Where: vd = velocity-depth; and

c = cover.

The quantity and diversity of cover available for the fish community at each site was graphically expressed in a stacked bar chart as the FHR for each vd class.

3.6.2. The Fish Response Assessment Index (FRAI)

The Fish Response Assessment Index (FRAI) (Kleynhans, 2007) was utilised in determining the integrity of the fish community in the vicinity of the Project area. The use of fish communities in the monitoring of aquatic ecosystems have been widely used to determine the overall condition of aquatic ecosystems. Fish communities have certain advantages when used as indicators of ecosystem integrity (NJDEP, 2000), namely:

Fish are present in most aquatic ecosystems unless the system is highly degraded; Fish can be easily identified and returned to the aquatic ecosystem; Most fish species have background information available in terms of life-history and environmental response data;

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Fish are mobile and may serve as good indicators of contamination and habitat degradation within a river reach; Fish are generally long-lived and can provide long-term information regarding environmental stress; Fish communities are composed of various trophic levels and can indicate stressor responses at many trophic levels; Fish often exhibit physiological, morphological or behavioural responses to stresses, which have been grouped into chemical stressors, physical stressors, and perceived stressors; and Due to the importance of safe fish consumption and recreational, subsistence and commercial fishing activities, the public is likely to relate to information concerning fish communities rather than other biotic communities.

The sampling of fish populations was undertaken in line with the FRAI methodology and the implementation manual for the River Health Programme (RHP), South Africa (Mangold, 2001). Fish were sampled within the habitat available at each site using the electroshocking technique (Meador et al., 1993; Barbour et al., 1999). This was carried out by employing a battery-operated electro-fishing apparatus for 35 minutes to 2 hours at each site depending on the extent of the site and the availability of habitat. Additional techniques were applied if necessary, including gill and cast netting. All fish sampled were identified and released back into the system if a sample was deemed unnecessary. Preservation with 96% ethanol was undertaken for fish that required off-site identification.

The FRAI is an assessment index based on the principle that the response of fish species to various stressors within an ecosystem will in turn lead to changes in the fish species assemblage from the reference condition. These stressors may result from changes in environmental conditions such as habitat integrity, as a result of driver changes, which include physico-chemical water quality, hydrological and geomorphological variations within the ecosystem. The FRAI makes use of the fish intolerance and preference database that was compiled in 2001 (Kleynhans, 2003) and the Fish Reference Frequency of Occurrence (FROC) database compiled by Kleynhans et al. (2007a). These databases include reference data for freshwater fish species which are integrated into the FRAI. The intolerance and preference attributes are categorised into metric groups with constituent metrics that relate to the environmental requirements and preferences of individual species. Each metric is weighted in terms of its importance for determining the Ecological Category under natural conditions for the specific river reach that is being surveyed (Kleynhans, 2007; Kleynhans et al., 2007b). The frequency of occurrence database provides reference information on the number of sites within a specific reach a certain species of

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fish has occurred in during a defined period. The index compares habitat preference, intolerance to habitat changes and present frequency of occurrence of a particular fish species to the reference frequency of occurrence in order to calculate the Ecological Category of the river reach.

As reference fish species lists for the Lilongwe River, including the Kamuzu Reservoir (as it is on the same system), and the Lisungwi River system could not be derived from the FROC database and because reference information on the frequency of occurrence of fish species within these systems is not available from existing literature, the data obtained during this wet season survey should be utilised as reference values for future surveys. This reference fish species list and fish frequency of occurrence data should therefore be entered into the FRAI index, along with the fish species sampled during future surveys and the fish frequency of occurrence data. The metric groups will then be ranked, rated and integrated to obtain an automated FRAI index value and Ecological Category which is based on the reference and current frequency of occurrence values and the relative intolerance and preference ratings. The classification of the Ecological Categories will be applied in line with the RHP as specified in Table 3-5, in addition to the EcoStatus A to F continuum approach (Figure 3-1) according to Kleynhans and Louw (2007) as this allows for boundaries to reflect artificially- defined points along the continuum to accommodate cases where there is uncertainty as to which category a particular entity belongs.

Table 3-5: Classification of Ecological Categories in line with the River Health Program

Ecological Category Description

A Unmodified, Natural.

B Largely Natural, with Few Modifications.

C Moderately Modified.

D Largely Modified.

E Extensively Modified.

F Critically Modified.

Figure 3-1: Ecological Categories according to the EcoStatus A to F Continuum Approach

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3.7. Mollusc Assessment

Species within the family Mollusca were sampled within the biotopes available at each site using a SASS net with a pore size of 1000 micron. The method included the kicking of rocky substrate in the stones biotope, the churning up of substrate in the gravel, sand and mud biotope and the prodding of marginal and aquatic vegetation from underneath the biotope to dislodge any individuals residing within these habitats. Once the biotope had been disturbed, the net was swept across the habitat several times in order to capture the molluscs within the net. All molluscs sampled were then identified and released back into the system.

3.8. Metal Analysis

Two fish species, namely, Astatotilapia calliptera and Oreochromis lidole, were selected for baseline tissue metal analysis as a result of the occurrence of these species at most of the sites. Two specimens were collected from each site where possible, in order to obtain a mean metal concentration for each species occurring at each site to ensure greater accuracy in the results. Two specimens of A. calliptera were collected at Sites MML1, MML3 and the Kamuzu Reservoir II, and two specimens of O. lidole were collected at Site MML1 and the Kamuzu Reservoir II. One specimen of A. calliptera was collected at Site MML2 and MML4, along with one specimen of O. lidole at Site MML4. A single live specimen of the native bivalve mollusc, Coelatura mossambicensis, was sampled from Site MML4 on the Lilongwe River and also analysed in order to provide an indication of baseline metal concentrations within the molluscs of the river reach. Bivalve molluscs (mussels) are sessile, filter-feeding organisms, able to accumulate within their tissues both organic and inorganic pollutants such as pesticides, hydrocarbons, metals, etc. present in the water column (Viarengo and Canesi, 1991). Bivalves are also able to tolerate high concentrations of these pollutants (Widdows and Donkin, 1992) and are highly exposed to environmental factors as their gills are in constant contact with the surrounding aquatic environment, occupy a large surface area and are in constant motion during the gaseous exchange and feeding processes (de Oliveira et al., 2008). Gastropod molluscs (snails), in contrast, are mobile and herbivorous and thus the level of metals in their soft tissues reflects the levels accumulated in the algae (and possibly other aquatic macrophytes) they feed on (Cubadda et al., 2001). Their exposure to pollutants is therefore not as comprehensive (Jakimska et al., 2011). As live bivalves were found to be sparse in the area during the time of assessment, only one sample could be analysed. In addition, sediment was sampled from the Kamuzu Reservoir II and analysed in order to provide an indication of metal concentrations within the sediment of the reservoir as it is likely to be one of the first

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receptors of potential contamination in terms of aquatic ecosystems. It is possible for metals to accumulate within the sediment biotope of this ecosystem to form sediment ‘sinks’ (Lloyd, 1992). When metals are adsorbed onto sediments, their positional- availability and bio-availability may be altered. These metals may then be remobilised into the water column by a number of mechanisms under certain conditions (Coetzee, 1993), with the most common being oxidation caused by physical and biological activities (Chapman et al., 1998). Benthic and other sediment-associated organisms may be negatively affected by these metals in both a lethal and sublethal manner. This in turn, directly or indirectly, affects other species such as fish, wildlife and humans by direct consumption or bioaccumulation through the food chain [United States Environmental Protection Agency (US EPA), 2001].

Whole tissue samples were dried at a temperature of 40°C in a dry oven for five to seven days depending on the size of the sample. The dried tissue samples were milled and digested with acid in a South African National Accreditation System (SANAS) accredited laboratory in South Africa. A distilled water extraction of the sediment sample was also conducted. The metal concentrations, excluding mercury, were measured by means of an Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) scan. An Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) scan was used to determine the concentration of dissolved mercury. The metals analysed for included aluminium (Al), arsenic (As), chromium (Cr), copper (Cu), iron (Fe), lead (Pb), manganese (Mn), cadmium (Cd), selenium (Se) and mercury (Hg).

The metal concentrations within the fish and mollusc tissue samples were determined and evaluated against the Quality Guidelines for Human Consumption [European Union (EU), 2006] and the Median International Standards for Trace Elements [California Environment Protection Agency (CEPA), 2000; Food and Agriculture Organization (FAO), 1983]. The concentrations within the sediment sample were evaluated against the Sediment Quality Guidelines (consensus-based) by MacDonald et al. (2000), the Canadian Council of Ministers of the Environment (CCME, 2001) and US EPA (2006). A limitation of this assessment is that guideline values have only been calculated for some parameters however, concentrations of these parameters recorded during future wet season surveys may be compared temporally in order to identify and monitor any increases in these metals over time.

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3.9. Diatom Analysis

Diatoms are the unicellular algal group most widely used as indicators of river and wetland health as they provide a rapid response to specific physico-chemical conditions in water and are often the first indication of change. The presence or absence of indicator taxa can be used to detect specific changes in environmental conditions such as eutrophication, organic enrichment, salinisation and changes in pH. They are therefore useful for providing an overall picture of trends within an aquatic system as they show an ecological memory of water quality over a period of time. Diatom laboratory procedures were carried out according to the methodology described by Taylor et al. (2005). Diatom samples were prepared for microscopy by using the hot hydrochloric acid (HCl) and potassium permanganate (KMnO4) method (Hasle, 1978; Taylor et al., 2007a). Approximately 300 to 400 diatom valves1 were identified and counted to produce semi-quantitative data for analysis as diatom counts of 300 valves and above are necessary to make correct environmental inferences (Prygiel et al., 2002). The taxonomic guide by Taylor et al. (2007b) and Cantonati et al. (2017) were consulted for identification purposes. Where necessary, Krammer and Lange-Bertalot (1986, 1988, 1991a and 1991b) were used for identification and confirmation of species identification. Environmental preferences were inferred from Taylor et al. (2007b), Cantonati et al. (2017) and various other literature sources as indicated in the discussion section, to describe the environmental water quality at each site (Ecotone, 2018).

The Specific Pollution Index (SPI) (CEMAGREF2, 1982) and the Biological Diatom Index (BDI) (Lenoir and Coste, 1996) were used in the diatom assessment and were calculated using Version 4.2 of the OMNIDIA software (Lecointe et al., 1993). The SPI is an inclusive index and takes factors such as salinity, eutrophication and organic pollution into account. This index comprises 2035 taxa (Taylor, 2004) and is recognised as the broadest species base of any index currently in use. It has been adapted to include taxa endemic to and commonly found in South Africa, thus increasing the accuracy of diatom-based water quality assessments and is known as the South African Diatom Index (SADI) (Harding and Taylor, 2011). Although it may not include taxa specific to Malawi, this index was used in the absence of any other country-specific indices. The limit values and associated ecological water quality classes used in this study to interpret the SPI and BDI scores are indicated in Table 3-6. The SPI and BDI are based on a score between 0 and 20, where a score of zero indicates an increasing level of pollution or eutrophication and a score of 20 indicates no pollution (Ecotone, 2018).

1 The siliceous unit that lies at each end of the frustule. The valve morphology is used to classify diatoms. 2 The Institut national de recherche en sciences et technologies pour l'environnement et l'agriculture (IRSTEA), formerly known as Cemagref, is a public research institute in France focusing on land management issues, such as water resources and agricultural technology.

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Table 3-6: Limit and Class Values used for Diatom Indices in the Evaluation of Water Quality [Adapted from Eloranta & Soininen (2002)]

Index Score Class >17 High quality 13 to 17 Good quality 9 to 13 Moderate quality 5 to 9 Poor quality <5 Bad quality

In addition, the Percentage of Pollution Tolerant Valves (%PTV) (Kelly and Whitton, 1995) and ecological descriptor indices (Van Dam et al., 1994) were also applied in this study to indicate possible impacts of organic pollution. The %PTV is part of the United Kingdom Trophic Diatom Index (TDI). The initial index by Kelly and Whitton (1995) was developed for monitoring possible impacts of organic pollution from sewage outfall (orthophosphate- phosphorus concentrations), and not general stream quality. The revised version of the TDI by Kelly (1998) made provision for the monitoring of general stream quality and has therefore been applied to this study. The %PTV has a maximum score of 100, where a score of zero indicates no organic pollution and a score of 100 indicates definite and severe organic pollution (Table 3-7). The presence of more than 21% PTVs indicates organic impact (Ecotone, 2018).

Table 3-7: Interpretation of the % PTV Scores (Adapted from Kelly, 1998)

% PTV Interpretation <20 Site free from organic pollution. 21 to 40 There is some evidence of organic pollution. Organic pollution likely to contribute significantly to 41 to 60 eutrophication. >61 Site is heavily contaminated with organic pollution.

3.10. Whole Effluent Toxicity (WET) Testing

The Direct Estimation of Ecological Effect Potential (DEEEP) method developed by the South African Department of Water Affairs and Forestry (DWAF, 2003) was utilised in assessing the ecological hazard of the surface water samples obtained from the biomonitoring sites. Acute toxicological screening was carried out by exposing biota to the water samples within environmental control rooms in order to determine the potential risk of the water to the integrity of the biota within the water body present at each site. This was conducted on biota representing four trophic levels, namely, Vibrio fischeri (bacteria), Selenastrum

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capricornutum (algae), Daphnia magna (macro-invertebrates) and Poecilia reticulata (fish) as test organisms. The Organisation for Economic Co-operation and Development (OECD) Guideline 201 (2006) was adhered to for the Selenastrum capricornutum growth inhibition test and the European Norm International Standards Organisation (EN ISO) 11348-3 (1998) was adhered to for the Vibrio fischeri bioluminescent test (Table 3-8). The US EPA (1993) protocol was performed for the Daphnia magna acute toxicity test and the US EPA (1996) protocol was performed for the Poecilia reticulata acute toxicity test (Table 3-9). Details on the sample preparation and methodologies for these protocols are summarised in the sections below.

3.10.1. Sample Preparation

The sample diluents used in the Vibrio fischeri bioluminescent test consisted of 20% mass/volume (m/v) sodium chloride (NaCl) stock solution. The diluent was used for sample preparation, dilution and control medium. The Vibrio fischeri bioluminescent screening test was performed with 100% of the sample and the definitive test consisted of a minimum of five sample concentrations selected to approximate a geometric series, i.e. 100%, 50%, 25%, 12.5% and 6.25% by using a dilution factor of 0.5.

Algal culturing medium used for the control and sample dilution was prepared with deionised water according to the OECD formula (OECD, 2006). The Selenastrum capricornutum growth inhibition screening test was performed with 100% of the sample and for the definitive test, and a 1:1 serial dilution was prepared with culturing medium.

Standard synthetic hard water (US EPA, 1993) was used as control and dilution medium for the Daphnia magna and Poecilia reticulata acute toxicity tests to establish their inherent toxicity (Slabbert, 2004). The Daphnia magna and Poecilia reticulata acute toxicity screening tests were performed with 100% of the sample and the definitive tests consisted of a minimum of five sample concentrations selected to approximate a geometric series. These test dilutions were prepared by serial dilution using a measuring cylinder.

3.10.2. Detailed Methodologies

Lyophilised3 Vibrio fischeri was reconstituted, left to stabilise at 150C for at least 1 hr, and then added to salinity-adjusted sample dilutions. The intensity of the luminescence of each sample was measured at T0, T15min and T30min. Bioluminescent inhibition was determined against control values (Table 3-8). Test validity was determined by a correction factor value between 0.6 and 1.8; replicate values that do not differ by more than 3%; and an

3 Biological substances dried by freezing in a high vacuum.

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appropriate inhibition value for the reference toxicant (20%-80% inhibition with 18.7mg/l hexavalent chromium (Cr6+).

Algal cultures in the exponential growth phase were used to provide an initial inoculum of 10 000 cells per long cell cuvette. Three 25ml replicates of each sample concentration were maintained at 250C with continuous lateral lighting (10 000 lux). The optical density (at 670 nanometre (nm) of the algae sample was recorded daily, and growth inhibition was determined relative to growth in the control (Table 3-8). Control growth needs to exceed a factor of 67 after 72 hours for the test to be valid. Algal culturing medium used for the controls and samples were prepared with deionised water according to the OECD formula (OECD, 2006). The Selenastrum capricornutum growth inhibition definitive test is performed with a 1:1 serial dilution series (e.g.100%, 50%, 25%, 12.5% and 6.25%).

Daphnia magna test organisms were cultured in the laboratory under specified conditions. Adult females were maintained in 200ml standard synthetic hard water (US EPA, 1993). Neonates (<24 hours old) were removed from adult female cultures, transferred to an intermediate holding container and then placed in test vessels containing control water or sample/dilutions. Twenty neonates (5 in each of 4 containers), were used per sample concentration. Mortality is recorded at 24 and 48 hours (Table 3-9). The test is valid if the control mortality is ≤10%. Daphnia magna acute toxicity test results were expressed in terms of percentage mortality for screening tests and as Lethal Concentration 10% (LC10) values and Lethal Concentration 50% (LC50) values for definitive tests performed.

Poecilia reticulata test organisms were cultured in the laboratory under standardised conditions. Test organisms less than 21 days old were transferred to the sample/sample dilutions and control (US EPA, 1996). The fish were exposed to the test sample for a period of 96 hours during which survival was monitored (Table 3-9). Mortality equal to or exceeding 10% in the screening tests indicated toxicity of the sample, provided mortality recorded in the control was equal or less than 10%. Results were expressed in terms of percentage mortality for screening tests and as LC10 values and LC50 values for definitive tests.

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Table 3-8: Summary of the Test Conditions and Test Acceptability Criteria for the Vibrio fischeri and Selenastrum capricornutum Acute Toxicity Screening Tests (Biotox, 2018)

Vibrio fischeri Bioluminescence Selenastrum capricornutum Aspect Test Growth Inhibition Test (EN ISO 11348-3, 1998) (OECD Guideline 201, 2006)

Standard Method SANS 11348-3:2013 SANS 8692:2015

Deviation None None

Test Species NRRL B-11177 Printz (CCAP 278/4 Cambridge, U.K)

Test Chamber N/A 10 cm long cell

Exposure Period 15 and 30 minutes 72hr Test Sample Volume 500 μl 25ml

Number of Replicates 2 3

Measurement Luminoscan TL, Hygiena Jenway 6300 spectrophotometer Equipment Monitoring System

Screening test - % growth Screening test - % growth inhibition Test Endpoint inhibition or stimulation relative or stimulation relative to control. to control; Definitive test - EC20 Definitive test - EC20 and EC50 and EC50 -values values

Statistical Method Biotox software (from supplier) – EXCEL spread sheet formulated by Used Normalized regression of relevant supplier (MicroBioTests Inc., data points Belgium) Algae batch number: SC141217 Matrix dissolving batch number: Batch Numbers / VF 171214 / 2020-05; RD 171214 / MD100118 Expiry Dates 2020-05; SD 171214 / 2020-03 Bead batch number: A-SC151116; B- SC151116; C-SC151116; D-SC151116

Test Validation 0.79 (valid if between 0,6 & 1,8) 79 (valid if cell density factor ≥67)

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Table 3-9: Summary of the Test Conditions and Test Acceptability Criteria for the Daphnia magna and Poecilia reticulata Acute Toxicity Screening Tests (Biotox, 2018)

Daphnia magna Acute Toxicity Poecilia reticulata Acute Test Toxicity Test Aspect (US EPA 600/4-90/027F, 1993) (US EPA 712-C-96-118, 1996) Standard Method SANS 6341:2015 OECD guideline 203 Deviation None None Test Species Daphnia magna Poecilia reticulata Test Species Age Less than 24h old Less than 21 days Exposure Period 24 and 48h 96hr Test Sample Volume 25 ml 200 ml Number of Test Organisms per 5 6 Well

Number of Replicates 4 2 Test Temperature 21±2˚C 21±2˚C

Test Endpoint Screening: % mortality Screening: % mortality Definitive: LC10 and LC50 Definitive: LC10 and LC50 values values

Statistical Method Used Trimmed Spearman Karber Trimmed Spearman Karber (TSK)/ Graphical interpolation (TSK) / Graphical calculated by linear regression interpolation calculated by of relevant data points, EXCEL relevant data points, EXCEL spread sheet spread sheet

Batch Numbers / Expiry Dates Ephippia - 290118; ISO control Control medium - 150118 relevant data points, EXCEL medium – 150118 spread sheet

Test validation 0% (valid if ≤10%) 8% control mortalities (valid if ≤10%)

For the Daphnia magna and Poecilia reticulata screening tests, a risk/hazard category was determined by application of a hazard classification according to the Direct Estimation of Ecological Effect Potential (DEEEP) method (Slabbert et al., 1998). This risk category equates to the level of acute/chronic risk posed by the receiving aquatic resource to these two trophic levels. These categories are indicated in Table 3-10.

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Table 3-10: Hazard Classification System for Daphnia magna and Poecilia reticulata Screening Tests

Percentage Mortality Class Description

None of the tests indicate a toxic effect. I No Acute Hazard

A statistically significant Percentage Effect (EP) is reached in at II Slight Acute Hazard least one test, but the effect level is below 50%.

The 50% EP (EP50) is reached or exceeded in at least one test, but III Acute Hazard the effect level is below 100%.

The 100% EP (EP100) is reached or exceeded in at least one test. IV High Acute Hazard

Very High Acute The 100% EP (EP100) is reached or exceeded in all tests. V Hazard

3.10.3. Toxicity Units

As toxicity involves an inverse relationship to effect concentrations4 (the lower the effect concentration, the higher the toxicity of an effluent), it may be presented more clearly if concentration-based acute toxicity measurements are translated into toxicity units (TUa). The major advantage of using toxicity units to express toxicity test results is that toxic units increase linearly as the toxicity of a sample increases. Toxic units also make it easy to specify water quality criteria based on toxicity.

The toxicity unit (TUa) for each test performed was calculated as 100% (sample) divided by the EC50 (e.g. 30 min Vibrio fischeri bioluminescent test and/or 72h Selenastrum capricornutum growth inhibition test) or LC50 (e.g. 48h Daphnia magna and/or 96h Poecilia reticulata acute toxicity test) values. Toxicity units are grouped as indicated in Table 3-11 (Tonkes and Baltus, 1997; DWAF, 2003).

Table 3-11: Grouping of Toxicity Units

Toxicity Unit Classification

<1 Limited to No Acute Toxicity

1-2 Negligible Acute Toxicity

2-10 Mildly Acute Toxicity

10-100 Acute Toxicity

>100 Highly Acute Toxicity

4 The concentration of the sample that causes a statistically significant sublethal effect on test organisms.

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4. RESULTS & DISCUSSION

The following results were derived from the aquatic biomonitoring assessment conducted during February 2018. A discussion of the results is also included within this section.

4.1.1. Environmental Context of the Project Area

The Project area is located approximately 15 km southwest of Lilongwe in Malawi. Other towns in close proximity to the Project area include:

Matsimbe - 3.3 km south of the Project area; Sinyala – 5 km southwest of the Project area; and Kaunda – 5 km northeast of the Project area.

4.1.1.1 Climate

The Project area is characterised by seasonal summer rainfall. The area has a temperate climate with hot wet summers and dry winters (Peel et al., 2007; TAHMO, 2017). The majority of the rainfall occurs from the months of November to April. A Mean Annual Precipitation (MAP) of 860 millimetres (mm) occurs in the area (TAHMO, 2017). According to the closest weather station at Sinyala, approximately 5 km from Malingunde, the MAP was approximately 914.6 mm, therefore receiving more rainfall in relation to the Project area. The rainfall variability within the region is a result of topographic irregularities that have an influence on the micro-spatial rainfall distribution (Reid, 2012).

4.1.1.2 Topography, Relief and Land Use

Lilongwe is located on a plateau in Central Malawi and forms part of the East African Rift Valley (Ebinger, 2005). The area is topographically irregular and is situated at an altitude of 1050 m above sea level (masl). The Project area is generally situated between 1100 and 1200 masl. Land use in the area mainly includes crop cultivation, urban and rural settlements, subsistence livestock farming and fishing.

4.1.1.3 Water Resource Areas (WRAs) and Water Resource Units (WRUs)

Malawi’s drainage system is divided into 17 Water Resources Areas (WRAs), which are subdivided into 78 Water Resources Units (WRUs) (UN-DTCV, 1986a). The Project area falls within the Linthipe Catchment WRA 4 and WRU 4D (Figure 4-1). The Lilongwe River subcatchment falls within the Linthipe River catchment area and is the largest of the subcatchments. The Lilongwe River flows from the Dzalanyama Mountain Range to the Linthipe River. The Kamuzu Reservoirs I and II regulate the flow in the Lilongwe River. Water flows from the Kamuzu Reservoir I to the Kamuzu Reservoir II after which it is released into the Lilongwe River as and when required. The Lilongwe River flows to a raw

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water intake weir at the waste water treatment plant in the city of Lilongwe, about 20 km downstream of the Kamuzu Reservoir II. This treated water is then abstracted to supply water to the community. However, 8% of the volume is discharged back into the Lilongwe River system in order to meet the Environmental Water Requirements (EWR) (Nemus, 2015).

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Figure 4-1: Water Resource and Major Catchment Areas in the vicinity of the Project Area

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4.1.1.4 Conservation Value of the Project Area with Regards to Freshwater Ecosystems

Aquatic ecosystems of ecological importance and sensitivity within or in close proximity to the Project area were assessed using the available literature. This information is summarised in the following points:

Protected wetlands under the Ramsar and United Nations (UN) biodiversity conventions include the major wetlands of Lake Malawi and Lake Chilwa, which are located 95 km to the east and 235 km south east of the Project area, respectively (Nemus, 2015); The wetlands (dambos) in the vicinity of the Project area (Figure 4-2) are an important resource to the local communities as these ecosystems guarantee food security, good grazing and hunting sites as well as water availability (Frenken and Mharapara, 2002); Important ecological services provided by the wetlands include flood assimilation, carbon storage, water temperature regulation and biodiversity maintenance (Nemus, 2015); Fauna such as Natriciteres olivacea (Olive Marsh Snake), Cisticola njombe (Churring Cisticola), Bugeranus carunculatus (Wattled Crane), Aonyx capensis (African Clawless Otter), Hydrictis maculicollis (Spotted-necked Otter) and Laephotis botswanae (Botswanan Long-eared Bat) have been historically documented to occur within the wetlands in Malawi and have a high potential to occur in the greater areas surrounding the Project area (Hughes and Hughes, 1992; Nemus, 2015); and Aonyx capensis and Hydrictis maculicollis are listed as Near Threatened on the International Union for Conservation of Nature (IUCN) Red List of Threatened Species, and Bugeranus carunculatus is listed as Vulnerable (IUCN Red List, 2018-2).

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Figure 4-2: Major Wetlands and Reserves in the vicinity of the Project Area

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4.2. Visual Survey

A visual survey of the eight sites was carried out during the wet season (high flow) assessment in February 2018. This was carried out in order to describe the state of the sites at the time of each assessment. Table 4-1 provides descriptions of each site, along with photographs depicting the up-and downstream features of the sites, where applicable.

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Table 4-1: Summary of Descriptions and Photographs of Each Site for the High Flow Survey in February 2018

Site Description Photo Upstream Photo Downstream

MML1 High, slow flows were evident at this point on the Lilongwe River during the high flow assessment. Water levels were high in the main channel and lower towards the bank edges. The site is characterised by a wide (>10m) channel, muddy substrate, aquatic macrophytes, grassy marginal vegetation and limited flow diversity. The riparian zone was dominated by grass. Trees and smaller shrubs were

scattered behind the grassy margins. Figure 4-3: Upstream view in a southerly direction Figure 4-4: Downstream view in a northerly direction Water was clear in the main channel indicating the high flow of the river at this point and indicating the slow flowing system at the time of and no algal growth was evident. the good cover provided by aquatic macrophytes. assessment. There is some potential for erosion on the left bank due to frequent livestock watering. Siltation is also likely to be occurring due to sediment build-up from the presence of the dam wall downstream. The site is also impacted by agricultural practices to the east. The Dzalanyama Forest Reserve occurs to the west of this site.

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Site Description Photo Upstream Photo Downstream

MML2 Mixed flow velocity was observed at this point on the Lisungwi River allowing for excellent variation in flow and depth, with shallow, faster runs occurring upstream and deeper, slower pools occurring downstream of this point. Excellent rocky substrate was available with stones ranging in size from 2 cm to 20 cm. An adequate amount of overhanging marginal vegetation

was present during high flow Figure 4-5: Upstream view in a south-easterly direction Figure 4-6: Downstream view in a north-westerly however, only reed stems and indicating fast runs occurring in the vicinity of the direction indicating the excellent stones biotope shoots were available for sampling bridge and flowing downstream. Overhanging marginal observed within a rapid which culminates in deep, as opposed to leafy vegetation vegetation in the form of reed stems is also evident. slow-flowing pools beyond this point. provided by the shrubs in the riparian zone. This limitation is due to the bank incision evident at this site likely resulting from fast flows scouring the banks during flood events and due to livestock watering. Water was silty and algal growth was evident on the rocks. This site is impacted by a road bridge and runoff from agricultural practices and rural settlements.

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Site Description Photo Upstream Photo Downstream

MML3 All three biotopes were sampled at this site during the current high flow survey. The stones and GSM biotopes were considered adequate however, overhanging marginal vegetation was limited. Although high water levels were observed at this site, some diversity in flow and depth occurred due to flow interception by the road bridge. Controlled releases from the

Kamuzu Reservoir located Figure 4-7: Upstream view in a south-westerly Figure 4-8: Downstream view in a north-easterly upstream of this point are likely to direction indicating the presence of deep pool habitat direction indicating the fast flow of the system and be contributing to the high water and the pillars of the road bridge. excellent bank cover present at this site. levels within the channel. The water was silty and clarity was poor. Algal growth was observed on the stones biotope. Excellent cover occurs on both banks reducing the risk of erosion. The channel is impacted on by a road bridge upstream of this point and runoff from surrounding agricultural practices and rural settlements.

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Site Description Photo Upstream Photo Downstream

MML4 The main channel at this site is wide (>5-10m) and deep (>1m). The system comprises run and pool habitats. Marginal vegetation and muddy substrate were present for sampling however, the stones biotope was unavailable. The overhanging vegetation is comprised of grass and some leafy vegetation. The water was silty at the time of survey and was flowing Figure 4-9: Upstream view in a southerly direction Figure 4-11: Downstream view in a north-easterly at a slow to medium flow velocity. indicating the high water levels observed within the direction indicating the excellent bank cover provided No algal growth was evident. Excellent vegetative cover occurs channel during the survey. by the marginal vegetation at this site. along the edges of both banks however, manual harvesting of river sand was also evident on both banks resulting in severe erosion and siltation of the system. This site is also impacted by livestock watering, runoff from agricultural practices and rural settlements, and controlled releases from the Kamuzu Reservoir resulting in increased water levels in the channel. Figure 4-10: Sand harvesting and trench formation Figure 4-12: Sand harvesting and extensive erosion of evident on the left bank of the channel. the right bank.

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Site Description Photo Upstream Photo Downstream

MML5 Similarly to Site MML4, the channel at this point of the Lilongwe River is wide (>5-10m) and deep (1m) and the system comprises run and pool habitats. The stones biotope was also unavailable for sampling. Marginal vegetation in the form of grass and reed stems was sampled both in and out of current and muddy substrate was abundant. The water was silty and slow-flowing.

Isolated clumps of algal growth Figure 4-13: Upstream view in a south-westerly Figure 4-14: Downstream view in a north-westerly were observed. Both banks have direction indicating the deep, slow-flowing run and direction indicating the incision and erosion of the left excellent vegetative cover excellent bank cover at this site. bank of the channel. however, incision and erosion are evident on the left bank due to frequent livestock watering leading to siltation of the system. This site is also impacted on by runoff from agricultural practices and rural settlements and controlled releases from the Kamuzu Reservoir which has resulted in increased water levels.

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Site Description Photo Upstream Photo Downstream

MMDR The narrow drainage line at this site consisted of very low water levels at the time of the survey. Both the stones and vegetation biotopes were unavailable for sampling during the survey. Muddy substrate was however prevalent. The water was discoloured and algal growth was evident. Channel modification, diversion and a road bridge impact on the flow of the

system. Bank erosion is evident as Figure 4-16: Downstream view in a southerly direction a result of the removal of indicating the stagnant water within the stream. indigenous vegetation. Alien invasive species were also noted. Water quality is impacted on by surrounding agricultural practices. The system is heavily impacted on by runoff from agricultural Figure 4-15: Upstream view in a northerly direction activities and rural settlements. indicating the low water levels occurring within the drainage line at the time of the assessment.

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Site Description Photo Upstream Photo Downstream

MMD1 The Kankoma dambo consists of a KAN shallow, stagnant, silty pool at the time of assessment. The vegetation and stones biotope were not available at this point. Muddy substrate was abundant. The water was silty and algal growth was observed. The site is impacted on by overgrazing from livestock farming resulting in the removal of indigenous vegetation and soil

erosion. The site is also impacted Figure 4-17: North-easterly view of the Kankoma Figure 4-18: North-easterly view of the Kankoma on by runoff from rural settlements dambo indicating the small, shallow pool observed dambo indicating the overgrazing of indigenous and agricultural practices. during the survey. vegetation by livestock resulting in soil erosion.

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Site Description Photo Upstream Photo Downstream

MMD2 The Kovuma dambo consists of a KO shallow, silty and stagnant pool with isolated clumps of algal growth. The water was found to be turbid. Marginal and aquatic vegetation were sampled along with muddy substrate. The stones biotope was, however, not available for sampling. Alien vegetation encroachment was observed at this site. Impacts

include flow modification as a result of a small road bridge, Figure 4-19: North-easterly view of the Kovuma dambo Figure 4-20: North-easterly view of the Kovuma dambo runoff from rural settlements and indicating the small, stagnant pool of silty water indicating the presence of marginal vegetation agricultural practices. containing aquatic macrophytes. alongside the road bridge that crosses the site.

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4.3. In Situ Water Quality Results

The in situ water quality measurements recorded during the aquatic biomonitoring investigation for the wet season (high flow) survey conducted in February 2018 are presented in Table 4-2.

Table 4-2: In situ Water Quality Results for the Wet Season Survey in February 2018

MMD1 MMD2 Parameter Guideline MML1 MML2 MML3 MML4 MML5 MMDR KAN KO 6.5 to 8.5 (WHO, 2011) pH <5% 7.17 7.64 8.11 7.87 8.24 6.38 7.1 6.78 Variation (DWAF, 1996) 5 to 30 Temp (oC) (DWAF, 26.3 26.9 27.5 27.9 30.8 25.8 27.5 26.1 1996) <150 (MBS, 2005) EC (mS/m) <15% 69 60 145 53 43 46 253 163 Variation (DWAF, 1996) <1000 TDS (mg/l) (MBS, 2005; 58.5 448.5 942.5 344.5 279.5 299 1644.5 1059.5 WHO, 2011) >5 mg/l (Kempster DO (mg/l) 7.0 7.8 7.5 7.3 7.2 5.6 6.2 6.4 et al., 1980)

The water quality measurements recorded at Sites MML1, MML2, MML4 and MML5 are considered to be fair during the current assessment. It is evident that the EC levels at Sites MML1 and MML2 have increased since the previous wet season survey undertaken in April 2017 (Figure 4-22). As the systems were in flood during the previous survey, it is likely that the nutrients were diluted resulting in the low EC values observed in April 2017. During the current survey however, these systems were not in flood, resulting in less dilution and higher readings. The increase in EC levels at Sites MML1 and MML2 are likely to have resulted from upstream sources on the Lilongwe and Lisungwi Rivers, respectively. These sources may include runoff containing nutrients such as nitrate, phosphate and ammonium ions from the surrounding villages and agricultural lands as well as from frequent livestock watering. Poor water quality conditions are evident at Site MML3, with a significant increase in EC level and an increase in the pH level; and at Sites MMDR, MMD1 KAN and MMD2 KO with an increase in

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EC levels (significant increases in EC levels have occurred at the two dambo sites) and a decrease in pH and DO levels occurring at these sites in relation to the previous survey (Figure 4-22; Figure 4-23; Figure 4-24). The increase in EC and pH levels at Site MML3 is likely due to the washing of garments with detergents by local communities upstream of this site, releasing phosphates into the system and resulting in an increase in the conductivity and the alkalinity of the water. In addition, inputs from runoff containing nutrients such as nitrate, phosphate and ammonium ions originating from the Lisungwi tributary and the villages and agricultural lands surrounding Site MML3 are also likely.

As mentioned, higher EC concentrations are evident at Sites MMDR, MMD1 KAN and MMD2 KO in relation to the previous survey. The EC levels at Sites MMD1 KAN and MMD2 KO exceed the MBS (2005) guideline for EC concentrations. The reason(s) for the significant increase in EC levels at these sites since the previous survey is likely due to lower water levels within these systems, coupled with runoff from surrounding villages and agricultural lands at these sites as well as the deposition of faeces and urine at Site MMD1 KAN from frequent livestock watering. The low pH level observed at Site MMDR may possibly be due to the nitrification of ammonium (conversion of ammonium to nitrate in soils by bacteria) likely to be present within these water bodies due to fertilizers containing ammonium such as urea, which is one of the fertilizers that is likely being applied to the agricultural lands in the vicinity of the sites [International Fertilizer Development Center (IFDC), 2013]. During the process of nitrification, hydrogen (H+) is released, increasing acidity. This is also supported by a decline in the DO level at this site. This is likely to limit the diversity and sensitivity of the aquatic macro-invertebrate communities that occur at these sites. The pH level recorded at Site MML5 is slightly alkaline, likely originating from the release of phosphate ions as a result of faecal deposition from frequent livestock watering practices occurring at this site. Temperature values fall within the guideline range of between 5 and 30°C at all sites with the exception of Site MML5, where the temperature guideline value is slightly exceeded and may limit the aquatic integrity at this point. Impacts resulting in the elevation of water temperature at this site are unknown at this stage. The DO level at all sites is above the 5 mg/l guideline value for the protection of aquatic biota (Kempster et al., 1980) and is therefore unlikely to limit the aquatic integrity at these points.

Spatially, the EC level increases by 110.1% between Sites MML1 and MML3 (Figure 4-21). As mentioned, the increase in EC level at Site MML3 may be due to the frequent washing of garments with detergents by the local community which was evident downstream of the Kamuzu Reservoir II wall. In addition, runoff containing nutrients such as nitrate, phosphate and ammonium ions from the surrounding villages and agricultural lands as well as from frequent livestock watering. Inputs from the Lisungwi tributary are also likely. This variation

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exceeds the guideline stipulating that the EC level along a watercourse should not vary by more than 15% and is likely to be affecting the aquatic communities at Site MML3 in the form of osmotic stress (DWAF, 1996). A 13.1% increase in the pH level is also observed between Sites MML1 and MML3 (Figure 4-21) which exceeds the guideline stipulating that the pH level along a watercourse should not vary by more than 5%. This is also likely due to garment washing, runoff from surrounding villages and agricultural lands, and frequent livestock watering.

Figure 4-21: Spatial Variation in Water Quality between the Monitoring Points on the Aquatic Resources in the vicinity of the Project Area in February 2018

The EC and pH levels between Sites MML2 and MML3 increase by 141.6% and 6.1%, respectively, indicating possible impacts on the Lilongwe River from the Lisungwi River system. A decrease of 63.4% and 2.9% in EC and pH levels, respectively, is evident between Sites MML3 and MML4, indicating the dilution and possible uptake of nutrients in the system by vegetation (Figure 4-21). Although the decrease in EC level may be viewed as an improvement towards more natural conditions, the variation in EC level between sites exceeds the guideline stipulating that the conductivity along a watercourse should not vary by more than 15%. A further decrease in EC levels of 70.3% and 18.8% is evident between Sites MML3 and MML5; and MML4 and MML5, respectively, which also exceeds the guideline. With regards to pH levels, a slight increase of 1.6% is evident between Sites MML3 and MML5 and an increase of 4.7% occurs between Sites MML4 and MML5, which falls within the guideline for spatial variations in pH level. However, in relation to the reference Site MML1,

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the EC levels at Sites MML4 and MML5 decrease by 23.1% and 37.6%, respectively, and the pH levels increase by 9.7% and 14.9%, respectively, exceeding the guidelines (Figure 4-21). An effect resulting from osmotic stress is therefore likely to be occurring on the aquatic community between these sites and future monitoring of these variations in EC and pH levels is therefore deemed essential.

Temporally, since the previous wet season survey in April 2017, the EC level at Site MML1 has increased by 1625% (17.25X) during the current assessment (Figure 4-22). As mentioned, the likely reason for the significant increase in EC level at the reference Site MML1 during the February 2018 survey is due to lower water levels as the system was not in flood at this time, resulting in less dilution and higher readings being recorded. Inputs from agricultural lands to the east of the site, local settlements to the south east and frequent livestock watering are likely to have contributed towards the increase in phosphates, nitrates and ammonium ions within the system, and thus the increase in the EC level at Site MML1. A significant increase in EC level of 233.3% (3.3X), 2800% (29X), 48.3% (1.4X), 2200% (21.3X) and 2560% (4.6X) is also evident at Sites MML2, MML3, MMDR, MMD1 KAN and MMD2 KO, respectively, since the previous survey (Figure 4-22). The degree of variation in the EC levels at all the sites therefore significantly exceed the guideline stipulating that the EC level should not vary seasonally by more than 15% (DWAF, 1996) and this is likely to be affecting the aquatic communities present in these systems at the current time as a result of osmotic stress.

Figure 4-22: Temporal Variation in Electrical Conductivity between the Wet Season Surveys undertaken in April 2017 and February 2018 at the Biomonitoring Sites

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The pH level has remained relatively stable at Site MML1, decreasing by 1.3% since the previous survey in April 2017, which falls within the guideline stipulating that the pH level should not vary seasonally by more than 5% (Figure 4-23). Similarly, the pH level at Site MML3 has increased by 3.9% which also falls within the guideline. However, the pH level at Site MML2 has increased by 5.9%, slightly exceeding the guideline, and has decreased at Sites MMDR, MMD1 KAN and MMD2 KO by 15.7%, 27.7% and 9.9%, respectively, also exceeding the guideline. Some impact due to temporal variations in pH levels is therefore expected to be occurring on the aquatic community at Sites MML2, MMDR, MMD1 KAN and MMD2 KO at this time.

Figure 4-23: Temporal Variation in pH between the Wet Season Surveys undertaken in April 2017 and February 2018 at the Biomonitoring Sites

Figure 4-24: Temporal Variation in Dissolved Oxygen between the Wet Season Surveys undertaken in April 2017 and February 2018 at the Biomonitoring Sites

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A decreasing trend in DO level is evident at Sites MML1, MML2, MML3, MMDR, MMD1 KAN and MMD2 KO since the previous survey in April 2017 (Figure 4-24). This may possibly be due to an increase in microbial activity and hence oxygen consumption as a result of higher concentrations of nutrients within these systems.

With regards to temperature, an increasing trend is evident at the above-mentioned sites since the previous survey (Figure 4-25). This is likely due to slightly higher average daily temperatures occurring in February in the Lilongwe area compared to April (Weatherspark, 2018).

Figure 4-25: Temporal Variation in Temperature between the Wet Season Surveys undertaken in April 2017 and February 2018 at the Biomonitoring Sites

4.4. Habitat Integrity

4.4.1. General Habitat Integrity

During the wet season assessment undertaken in February 2018, the IHIA results in Table 4-3 indicate that the general habitat integrity may be regarded as being Moderately Modified (Class C) at Sites MML1, MML2 and MMD1 KAN, Largely Modified (Class D) at Sites MML3 and MML5 and Extensively Modified (Class E) at Sites MML4, MMDR and MMD2 KO. Data from the wet season survey conducted in April 2017 is presented in Table 4-4 for temporal trend analysis.

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Table 4-3: IHIA Results for the Wet (High Flow) Season in February 2018

MMD1 MMD2 IHIA Scores MML1 MML2 MML3 MML4 MML5 MMDR KAN KO Instream Score (%) 69.7 72.4 47.4 42.8 42.7 27.7 63.6 36.1 Riparian Score (%) 84.5 72.5 61.1 16.9 38.9 14.3 84.5 36.6 Overall Score (%) 77.2 72.5 54.3 29.9 40.9 21.0 74.1 36.4 Class C C D E D E C E

Since the previous wet season survey conducted in April 2017 (Table 4-4), the general habitat integrity has remained in a Moderately Modified (Class C) condition at Sites MML1 and MML2, has declined from a Largely Natural with Few Modifications (Class B) condition to a Largely Modified (Class D) condition at Site MML3, has declined from an Unmodified, Natural (Class A) condition to a Moderately Modified (Class C) condition at Site MMD1 KAN and from a Largely Modified (Class D) condition to an Extensively Modified (Class E) condition at Site MMD2 KO. The habitat integrity at Site MMDR has however improved from a Critically Modified (Class F) condition in April 2017 to an Extensively Modified (Class E) condition during the current survey (Table 4-3). The decline in the overall scores obtained at Sites MML1, MML2, MML3, MMD1 KAN and MMD2 KO is mainly a result of increased water quality modifications due to significant increases in EC levels (Figure 4-22).

Table 4-4: IHIA Results for the Wet (High Flow) Season in April 2017

MMD1 MMD2 IHIA Scores MML1 MML2 MML3 MMDR KAN KO Instream Score (%) 79.0 79.0 81.3 18.5 96.8 51.2 Riparian Score (%) 80.7 80.1 79.6 5.07 97.6 34.1 Overall Score 79.9 79.6 80.5 11.8 97.2 42.7 Class C C B F A D

During the current survey, Site MML1 on the Lilongwe River obtained a score of 69.7% for instream habitat integrity and 84.5% for riparian habitat integrity, with an overall IHIA score of 77.2% resulting in a Class C (Table 4-3). Impacts on instream habitat integrity at Site MML1 include small impacts as a result of flow, bed and channel modifications, moderate impacts due to inundation and the establishment of exotic macrophytes, and large impacts due to water quality modifications and solid waste disposal resulting from frequent livestock watering. Within the riparian zone, limited impacts have occurred due to indigenous vegetation removal, exotic vegetation encroachment, flow modifications and inundation during the current survey. Moderate impacts have however occurred on the left bank due to erosion resulting from livestock watering activities practiced on a frequent basis. This disturbance has in turn led to a moderate impact on water quality within the riparian zone.

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Small instream impacts due to water abstraction, flow, bed and channel modifications were evident at Site MML2 on the Lisungwi River, as well as moderate impacts due to solid waste disposal from livestock watering activities and a large impact due to water quality modifications. This system is not impacted by water releases from the Kamuzu Reservoir as it is a tributary of the Lilongwe River and therefore inundation of this system is limited. Impacts on the channel and flow within the riparian zone at this point due to the road bridge were limited. The riparian zone has also been moderately impacted on by indigenous vegetation removal and bank erosion. A large impact is however likely to be occurring due to water quality modification. Site MML2 obtained a score of 72.4% for instream habitat integrity and 72.5% for riparian habitat integrity, with an overall IHIA score of 72.5% resulting in a Class C (Table 4-3).

Site MML3 on the Lilongwe River obtained a score of 47.4% for instream habitat integrity and 61.1% for riparian habitat integrity, with an overall IHIA score of 54.3% resulting in a Class D. Instream impacts include small impacts on the channel and bed of the river resulting from the presence of a road bridge and associated pillars. The site is also largely impacted by water releases from the Kamuzu Reservoir resulting in large impacts on flow. Water quality modifications were also found to be severely impacting the site at this time. The presence of the road bridge and pillars within the riparian zone has resulted in a limited impact on the banks of the channel. Soil erosion is also limited at this point due to bank stabilisation by riparian vegetation. Moderate impacts have however occurred due to indigenous vegetation removal, exotic vegetation encroachment, water releases from the Kamuzu Reservoir and inundation. The site has also been largely impacted on by water quality modifications.

Site MML4 on the Lilongwe River obtained a score of 42.8% for instream habitat integrity and 16.9% for riparian habitat integrity, with an overall IHIA score of 29.9% resulting in a Class E (Table 4-3). Small instream impacts have occurred at Site MML4 as a result of solid waste disposal from livestock watering activities, along with a moderate impact on water quality and large impacts due to water releases, flow, bed and channel modifications and inundation. Severe impacts relating to the manual harvesting of river sand by local communities are evident within the riparian zone at Site MML4. This includes indigenous vegetation removal and channel modification which have subsequently led to large impacts on the river banks including erosion, incision and channel widening. This has caused a large amount of silt to enter the instream channel resulting in a lack of exposure of rocky substrate which is required by many macro-invertebrate species for survival. Further large impacts due to sand harvesting activities at the site include inundation and flow modifications within the riparian zone. This is evident in the significantly large pools of water that have formed within this zone over time resulting in instability and a high

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potential for bank collapse. The formation of several trenches was also evident on both banks. The riparian habitat is also moderately impacted by exotic vegetation encroachment and water quality modifications.

Site MML5 on the Lilongwe River obtained a score of 42.7% for instream habitat integrity and 38.9% for riparian habitat integrity, with an overall IHIA score of 40.9% resulting in a Class D. Instream impacts include moderate impacts due to water quality modifications and solid waste disposal from frequent livestock watering, and large impacts resulting from water releases, flow, bed, channel modifications and inundation. High water levels were therefore evident at Sites MML3, MML4 and MML5 as a result of the systematic release of water into the Lilongwe River system from the Kamuzu Reservoir. The riparian zone at Site MML5 is moderately impacted on by indigenous vegetation removal, exotic vegetation encroachment and water quality modifications, along with large impacts on flow due to channel modifications as a result of bank incision and erosion caused by frequent livestock watering. This has also led to the widening of the channel at this point and the deposition of large amounts of silt within the channel resulting in the lack of exposure of rocky substrate at this site as observed at Site MML4.

The instream habitat at Site MMDR has been largely impacted on by water abstraction for irrigation purposes, water quality modifications, the presence of exotic macrophytes, bed and channel modifications due to cultivation activities and the presence of the road bridge crossing the site. Moderate impacts as a result of flow modifications due to the canalisation of the system were also noted. The riparian zone has been largely impacted by indigenous vegetation removal, exotic vegetation encroachment, water abstraction, flow and channel modifications. Moderate impacts due to bank erosion and inundation were also evident within this zone. Site MMDR obtained a score of 27.7% for instream habitat integrity and 14.3% for riparian habitat integrity, with an overall IHIA score of 21.0% resulting in a Class E.

Small instream impacts have occurred at Site MMD1 KAN as a result of water abstraction, bed modification and the presence of exotic macrophytes. A moderate impact has occurred due to solid waste disposal from frequent livestock watering at the site and a critical impact has occurred as a result of water quality modifications. A moderate impact resulting from indigenous vegetation removal due to overgrazing by livestock was evident within the riparian zone. This has resulted in moderate levels of soil erosion and the encroachment of exotic vegetation. This site obtained a score of 63.6% for instream habitat integrity and 84.5% for riparian habitat integrity, with an overall IHIA score of 74.1% resulting in a Class C (Table 4-3). The water quality at Site MMD2 KO has been severely impacted upon during the current survey as a result of runoff from agricultural practices and rural settlements in the vicinity of the site. Severe flow modifications have also occurred as a result of the presence

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of a road bridge at the site and the canalisation of the system. The site has also been moderately impacted on by water abstraction for irrigation purposes, bed and channel modifications, as well as exotic macrophyte encroachment due to the disturbance. The riparian zone has been largely impacted on by indigenous vegetation removal, exotic vegetation encroachment, flow and water quality modifications. Moderate impacts by water abstraction, channel modifications, soil erosion and inundation were also evident. Site MMD2 KO obtained a score of 36.1% for instream habitat integrity and 36.6% for riparian habitat integrity, with an overall IHIA score of 36.4% resulting in a Class E.

4.4.2. Habitat Suitability

The integrity of the instream and riparian habitat has a direct influence on the structure of the aquatic community. The IHAS index was used to determine the suitability of the habitat, specifically for the requirements of the macro-invertebrate community.

The contribution of each biotope, namely Stones In Current (SIC), Vegetation (VEG), Other Habitat including Gravel, Sand and Mud (GSM), as well as the physical condition of the stream are calculated in order to work out the final IHAS score which reflects the overall habitat integrity of the site. This allows for the identification of each biotope from most to least prevalent. The results of the IHAS assessment conducted at the biomonitoring sites during the current wet (high flow) season survey in February 2018 are presented in Table 4-5. Data from the wet season survey conducted in April 2017 is presented in Table 4-6 for temporal trend analysis.

Table 4-5: IHAS Results for the Wet (High Flow) Season in February 2018

MMD1 MMD2 IHAS Biotopes Assessed MML1 MML2 MML3 MML4 MML5 MMDR KAN KO Stones In Current (SIC) 0 17 14 0 0 0 0 0 Vegetation (VEG) 15 10 9 8 9 0 0 12 Gravel, Sand & Mud (GSM) 12 16 11 12 9 9 9 11 Physical Stream Condition 27 28 25 22 20 21 18 21 Total Habitat Score 27 43 34 20 18 9 9 23 Total IHAS Score (%) 54 71 59 42 38 30 27 44

The IHAS results obtained during the current survey indicate that Site MML2 on the Lisungwi River is the only site that is considered adequate in supporting a diverse macro-invertebrate community. This site obtained the highest score with regards to habitat suitability required for the colonisation of the macro-invertebrate community during the wet season survey (Table 4-5).

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The habitat integrity at the remainder of the Sites MML1, MML3, MML4, MML5, MMDR, MMD1 KAN and MMD2 KO was however, found to be inadequate in supporting a diverse macro- invertebrate community. The habitat conditions on the Lilongwe River indicate some variation in habitat integrity between the sites, increasing in suitability from MML1 to MML3, after which a decreasing trend is evident further downstream at Sites MML4 and MML5 (Figure 4-26). This is likely to have an influence on the structure of the aquatic communities present at these sites.

The habitat suitability at Site MML1 has increased by 14.8% since the previous wet season survey undertaken in April 2017 (Figure 4-27). Similar to the previous wet season survey, the stones biotope at Site MML1 was absent during the current survey. This is likely to have resulted from the siltation of the channel due to bank incision and erosion caused by frequent livestock watering as well as due to sediment build-up resulting from the presence of the downstream dam wall, which has likely inhibited sediment transport by the flow regime (Freeman and Rowntree, 2005). However, the availability of marginal vegetation in the form of aquatic macrophytes and leafy vegetation was found to be more abundant during this survey. Muddy substrate was abundant at this point as was observed during the previous survey and the water was clear. Less algal growth was observed during this survey, therefore resulting in an increase in the GSM score (Table 4-5; Table 4-6). The system was also slow-flowing and deep during the current survey resulting in limited diversity in flow and depth profiles. This is likely to limit the diversity of the aquatic macro-invertebrate community at this point. The habitat at Site MML2 consisted of all three biotopes, namely stones, GSM and vegetation, and the habitat integrity at this site is considered adequate in supporting a diverse macro-invertebrate community. The integrity at this site has increased by 31.4% since the previous wet season survey (Figure 4-28). Similar to the previous survey, large cobbles, boulders and small pebbles were sampled in and out of current, along with more marginal vegetation than previously sampled. An adequate amount of overhanging marginal vegetation was present, however, it mainly consisted of stems and shoots, providing limited cover for macro-invertebrates. Sand, gravel and mud deposits were abundant, and the system consisted of fast-flowing rapids and moderately-fast flowing pools and runs. The water was silty and algal growth was evident on the rocky substrate. There was a good diversity in depth and flow. The riparian vegetation consists of a mix of grasses, shrubs and reeds, with a good amount of vegetation covering both banks, however, impacts in the form of bank erosion and incision are increasing at this point over time due to livestock watering as well as the frequent use of footpaths by the local community for activities such as fishing and bathing within the channel.

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Deep, fast-flowing water occurred at Site MML3 which is likely due to releases from the Kamuzu Reservoir. Although high water levels were observed at the site, some diversity in flow and depth occurred due to flow interception by the road bridge pillars. The habitat suitability at Site MML3 has however decreased by 14.4% since April 2017 (Figure 4-29). This is due to less availability of sandy substrate and overhanging marginal vegetation, particularly with regards to the absence of any leafy vegetation in relation to the previous survey. Impacts on bank cover were also evident as a result of the removal of indigenous vegetation from the left bank. However, the amount of cover present still allows for good bank stability minimising the potential for erosion at this site.

Table 4-6: IHAS Results for the Wet (High Flow) Season in April 2017

MMD1 MMD2 IHAS Biotopes Assessed MML1 MML2 MML3 MMDR KAN KO Stones In Current (SIC) 0 18 14 0 0 0 Vegetation (VEG) 13 9 11 0 10 11 Gravel, Sand & Mud (GSM) 7 8 15 2 10 9 Physical Stream Condition 27 19 29 28 21 21 Total Habitat Score 7 35 14 14 42 10 Total IHAS Score (%) 47 54 69 30 41 41

The habitat integrity at Sites MML4 and MML5 is inadequate in supporting a diverse macro- invertebrate community. This is mainly due to the high water level present at both sites resulting in limited depth and flow diversity which is likely a result of the controlled releases from the Kamuzu Reservoir. In addition, the gradual siltation of the channel from soil erosion due to extensive sand harvesting activities at Site MML4 and the incision of the banks caused by livestock watering at Site MML5, has resulted in the lack of rocky substrate exposure at both sites during the current survey. Only one to two meters of overhanging vegetation was sampled due to limited access to the banks as a result of the high water levels and the presence of abundant soft muddy substrate. The water was silty at both sites and isolated clumps of algal growth were evident at Site MML5.

The habitat integrity at Site MMDR has remained constant since the previous wet season survey (Figure 4-30). Only the GSM biotope was available for sampling at Site MMDR. The narrow, canalised stream was very shallow at the time of survey and muddy substrate dominated the site at this point. Isolated clumps of algae were evident and the water was discoloured. The riparian vegetation consisted of grasses, shrubs and some small reeds. Plantations occur upstream of this point, replacing most of the indigenous vegetation and

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giving rise to soil erosion. The site is also impacted by runoff from these plantations and the surrounding local settlements.

Sampling at Site MMD1 KAN was also restricted to the GSM biotope during the current survey resulting in a significant decrease in habitat integrity by 34.1% since April 2017 (Figure 4-31). This is likely due to the significant decrease in the volume of water and the lack of overhanging marginal and aquatic vegetation during the current survey due to overgrazing by livestock. Muddy deposits dominated the site within the shallow, stagnant pool and isolated clumps of algal growth were evident. The water clarity was poor as a result of the suspension of silt in the water from livestock watering activity. Riparian vegetation consisted of grass, however bare areas were present where the grass was heavily overgrazed, resulting in soil erosion.

The habitat integrity at Site MMD2 KO was also found to be inadequate in supporting a diverse macro-invertebrate community. However, the integrity of the habitat at the site has increased by 7.3% since April 2017 (Figure 4-32). Both marginal and aquatic vegetation were sampled during this survey, most of which was considered leafy. Muddy substrate was abundant. Algal growth was isolated and the water was silty. Runoff from agriculture and the surrounding local settlements impact on this site.

4.5. Aquatic Macro-Invertebrate Community Integrity Assessment

The results of the macro-invertebrate integrity assessment conducted at Sites MML1, MML2, MML3, MML4, MML5, MMDR, MMD1 KAN and MMD2 KO using the SASS5 index during the wet (high flow) season are presented in Table 4-7 and Figure 4-26. The PES class was determined by comparing the scores attained at each site to the reference conditions for sandy and rocky systems according to Table 3-2 (Tambala et al., 2016). Sites MMD1 KAN and MMD2 KO within the dambos are considered sandy as these systems lack rocky substrate, whilst Sites MML1, MML3, MML4 and MML5 on the Lilongwe River, Site MML2 on the Lisungwi River and Site MMDR on the drainage line are considered to be rocky systems. Data from the wet season survey conducted in April 2017 is presented in Table 4-8 for temporal trend analysis. Temporal variation in SASS5, IHAS and ASPT scores obtained during the wet season surveys conducted in April 2017 and February 2018 is also indicated for each site from Figure 4-27 to Figure 4-32, with the exception of Sites MML4 and MML5 as there is currently no temporal data available for these additional sites.

According to the results obtained, the macro-invertebrate integrity is regarded as being in a Severely to Critically Modified (Class E/F) state at Sites MML1, MML4, MML5, MMDR, MMD1

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KAN and MMD2 KO and in a Largely Modified (Class D) state at Sites MML2 and MML3 during the wet season survey. Site MML1 obtained the highest SASS5 score, followed by Sites MML2, MML3, MML4, MMD2 KO, MMD1 KAN, MML5 and lastly, Site MMDR. It is evident that the SASS5 score decreases in a downstream direction on the Lilongwe River indicating a decrease in macro-invertebrate diversity from Site MML1 to MML5 (Table 4-7; Figure 4-26). This is likely a result of a decline in habitat diversity downstream of the Kamuzu Reservoir on the Lilongwe River, likely due to the inundation of this system as a result of water releases from the reservoir. The inundation of the system prevents the establishment of many invertebrate taxa with a preference for shallow riffle, rapid and run habitats. Furthermore, siltation of the instream channel at Sites MML4 and MML5 has resulted in the lack of rocky substrate exposure due to significant bank erosion from manual sand harvesting activities at Site MML4 and livestock watering activities at Site MML5. With regards to the ASPT score, Site MML2 on the Lisungwi River attained the highest score, followed by Sites MML3, MML1, MML4, MMD1 KAN, MML5, MMD2 KO and MMDR. Site MMDR obtained the lowest ASPT score of 3.4, reflecting the presence of communities with the least sensitivity i.e. hardy, tolerant species at this site (Table 4-7; Figure 4-26).

Since the previous survey conducted in April 2017 (Table 4-8), the macro-invertebrate integrity has remained in a Severely to Critically Modified (Class E/F) state at Site MML1 (Table 4-7). At Site MML2, an improvement in the integrity is observed, from a Severely to Critically Modified (Class E/F) state in April 2017 to Largely Modified (Class D) state during the current survey. A decline in the integrity is however, evident at Site MML3, from a Moderately Modified (Class C) state in April 2017 to a Largely Modified (Class D) state during the current survey. The integrity at Site MMDR has remained in a Severely to Critically Modified (Class E/F) state, however a decline in condition is evident at Sites MMD1 KAN and MMD2 KO, from a Largely Modified (Class D) state during the previous survey to a Severely to Critically Modified (Class E/F) state during the current survey.

Table 4-7: SASS5 Results for the Wet (High Flow) Season in February 2018

MMD1 MMD2 Aspect MML1 MML2 MML3 MML4 MML5 MMDR KAN KO SASS5 Score 88 83 58 58 31 24 46 50 Number of Taxa 19 14 10 14 8 7 11 13 ASPT Score 4.6 5.9 5.8 4.1 3.8 3.4 4.1 3.8 PES Class E/F D D E/F E/F E/F E/F E/F

During the current survey, spatial results indicate a 51.7% decrease in the SASS5 score at Site MML3 in relation to the score obtained at Site MML1, reflecting higher diversity within the macro-invertebrate community at Site MML1 despite the lower IHAS score obtained at this

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site (Figure 4-26). This is likely a result of the presence of more abundant, leafy vegetation at Site MML1 compared to the reed stems present at Site MML3 that provide less suitable habitat for the macro-invertebrates compared to leafy vegetation (Table 4-5). Leafy vegetation was also found to be more abundant during the current survey at Site MML1 in relation to the vegetation available during the survey conducted in April 2017. However, a 20.6% increase in ASPT score is evident between Sites MML1 and MML3 (Figure 4-26), indicating the occurrence of more sensitive taxa at Site MML3 compared to Site MML1. This is unlikely to be a result of better water quality at Site MML3 as a 110.1% increase in EC level and a 13.1% increase in the pH level is evident between Sites MML1 and MML3 (Figure 4-21). The higher ASPT score at Site MML3 is more likely to be due to the presence of the stones biotope at this site which is absent at Site MML1 (Table 4-5). This biotope provides habitat for sensitive macro-invertebrate species from the families Ephemeroptera and Trichoptera, most of which have a strong affinity for rocky substrate. The lack of this biotope at Site MML1 has prevented the establishment of these sensitive species at this point thus resulting in a lower ASPT score. As mentioned, this is likely to have resulted from the siltation of the channel caused by erosion from frequent livestock watering and as a result of sediment build-up from the presence of the downstream dam wall that is likely inhibiting the transport of sediment by the flow regime (Freeman and Rowntree, 2005). The deposition of faeces within the channel at Site MML1, as well as runoff containing nutrients such as nitrate, phosphate and ammonium ions entering the channel from the surrounding villages and agricultural lands is also likely to have impacted on the water quality, reflected in the lower ASPT score. Therefore, a lower ASPT score at Site MML3 in relation to the ASPT score at Site MML1 is likely to be considered an indicator of significant impact on the water quality of the Lilongwe system downstream of the Project area for future monitoring. Both the SASS5 and ASPT scores decrease by 43.1% and 1.7%, respectively, between Sites MML2 and MML3 (Figure 4-26).

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Figure 4-26: Spatial Variation in SASS5, IHAS and ASPT Scores between the Monitoring Points on the Aquatic Resources in the vicinity of the Project Area in February 2018

The decrease in SASS5 score at Site MML3 indicates a lower diversity in macro-invertebrate community at this site on the Lilongwe River in relation to Site MML2 on the Lisungwi River. This is likely a result of less habitat availability and diversity at Site MML3 resulting from impacts related to the water releases from the Kamuzu Reservoir. As the habitat at Site MML2 on the Lisungwi River is not subjected to these releases, there is more diversity in depth and flow resulting in better habitat conditions for macro-invertebrates and hence a higher IHAS and SASS5 score at this site (Figure 4-26). The slightly lower ASPT score at Site MML3 in relation to the score obtained at Site MML2 indicates a less sensitive community at this site, which is likely a due to the elevated EC level recorded at this point during the current survey, along with a higher pH level in relation to the level recorded at Site MML2 (Figure 4-21). As mentioned in Section 4.3, this is likely due to the washing of garments using detergents by local communities upstream of Site MML3 and inputs from runoff containing nitrate, phosphate and ammonium ions originating from the Lisungwi tributary and the villages and agricultural lands surrounding Site MML3.

The SASS5 score obtained at Site MML4 remains consistent with the score obtained at Site MML3 (Figure 4-26). The score therefore also decreases by 51.7% in relation to the reference Site MML1, indicating a decline in macro-invertebrate diversity downstream of the reference site. The SASS5 score reflects similar community diversity at Site MML3, however, the stones biotope is lacking at Site MML4, preventing the establishment of sensitive macro- invertebrate species from the families Ephemeroptera and Trichoptera, likely resulting in

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the 41.4% decrease in ASPT score in relation to Site MML3. The ASPT score at Site MML4 also decreases by 12.1% in relation to Site MML1, which may also be attributed to the lower IHAS score attained at this site as a result of poor habitat diversity and suitability for macro- invertebrate species (Figure 4-26). This is mainly due to the inundation caused by the releases from the Kamuzu Reservoir resulting in poor diversity in depth and flow, as well as the siltation of the instream channel caused by the significant erosion arising from manual sand harvesting from the river banks and the livestock watering at this point, which has subsequently resulted in the lack of rocky substrate exposure.

The SASS5 and ASPT scores decrease by 183.8% and 21%, respectively, between Sites MML1 and MML5. A decrease in SASS5 score of 87% in relation to Sites MML3 and MML4 is also evident at Site MML5, along with a decrease in ASPT score of 52.6% and 7.8%, respectively. The decrease in SASS5 score is likely a result of poor habitat diversity and suitability which is reflected in the low IHAS score obtained at this site (Figure 4-26). The habitat at this site is also impacted by inundation and siltation as mentioned above. The banks at this site have been significantly incised as a result of livestock watering causing widening of the channel and the deposition of large amounts of sediment on the river bed resulting in the lack of exposure of rocky substrate. The decrease in ASPT score is likely to be a result of an increase in the pH level and elevated temperature levels at this site (Figure 4-21), along with the lack of the stones biotope. The increase in pH level is likely due to the release of phosphate ions as a result of faecal deposition from frequent livestock watering practices occurring at this site and runoff from surrounding agricultural practices and rural settlements. Impacts resulting in the elevation of water temperature at this site are however unknown at this stage.

Table 4-8: SASS5 Results for the Wet (High Flow) Season in April 2017

MMD1 MMD2 Aspect MML1 MML2 MML3 MMDR KAN KO SASS5 Score 49 44 74 15 71 54 Number of Taxa 12 9 11 3 15 12 ASPT Score 4.1 4.9 6.7 5.0 4.7 4.5 PES Class E/F E/F C E/F D D

Temporally, the SASS5 and ASPT scores obtained at Site MML1 during the current survey have increased by 79.5% and 12.1%, respectively, since the previous wet season survey conducted in April 2017. The increase in SASS5 score indicates an improvement in the diversity of the macro-invertebrate community and is likely a result of improved habitat conditions reflected in the increase in the IHAS score during the current assessment (Figure 4-27).

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As mentioned, the vegetation at this point was found to be more abundant during the current survey in relation to the survey conducted in April 2017. The increase in ASPT score is likely a result of the increase in the concentration of nutrients within the system as mentioned in Section 4.3. As the system was in flood during the previous survey, it is likely that the nutrients were diluted resulting in the lower EC values observed in April 2017 (Figure 4-27).

At Site MML2, an increase in both the SASS5 and ASPT scores have occurred since April 2017 (Figure 4-28). These scores have increased by 88.6% and 20.4%, respectively, indicating an improvement in both the diversity and sensitivity of the macro-invertebrate community at this point. The higher IHAS score indicates better habitat conditions during the current survey which is likely a result of a reduction in flow velocity and water levels as opposed to the flood conditions observed during April 2017. This has resulted in an increase in macro- invertebrate community diversity and thus the SASS5 score. The increase in the sensitivity of the community reflected in the increase in the ASPT score is also likely a result of the increase in the concentration of nutrients within the system as well as better habitat conditions.

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Figure 4-28: Temporal Variation in SASS5, IHAS and ASPT Scores obtained during the Wet Season Surveys undertaken in April 2017 and February 2018 at Site MML2. The Variation in EC and pH Levels are also indicated for ease of reference

Figure 4-29 indicates that both the SASS5 and ASPT score at Site MML3 have decreased by 21.6% and 13.4%, respectively, since April 2017. The decrease in SASS5 score is likely due to a decline in habitat suitability reflected in the lower IHAS score obtained during the current survey, specifically with regards to vegetation, GSM and physical stream condition (Table 4-5). As mentioned in Section 4.4.2, this relates to the decrease in the availability of sandy substrate and overhanging marginal vegetation, particularly leafy vegetation, and bank cover as a result of the indigenous vegetation removal. The decrease in ASPT score is likely due to the significant temporal increase in EC level observed at this point during the current survey which is likely to have resulted in osmotic stress within the aquatic macro- invertebrate community over time (Figure 4-29).

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Figure 4-29: Temporal Variation in SASS5, IHAS and ASPT Scores obtained during the Wet Season Surveys undertaken in April 2017 and February 2018 at Site MML3. The Variation in EC and pH Levels are also indicated for ease of reference

The SASS5 score at Site MMDR has increased by 60% whilst the ASPT score has decreased by 32% since the previous survey (Figure 4-30). The increase in the SASS5 score indicates an improvement in community diversity which is likely due to the presence of more muddy substrate and less algal growth evident during the current assessment (Table 4-5 and Table 4-6). The decrease in ASPT score indicates that the sensitivity of the community has declined since April 2017 which is likely due to the 48.3% increase in EC level and the low pH level of 6.38 (Figure 4-30).

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Figure 4-30: Temporal Variation in SASS5, IHAS and ASPT Scores obtained during the Wet Season Surveys undertaken in April 2017 and February 2018 at Site MMDR. The Variation in EC and pH Levels are also indicated for ease of reference

At Site MMD1 KAN, both the SASS5 and ASPT scores have decreased by 35.2% and 12.7%, respectively, since the previous survey. The SASS5 score is likely to have decreased as a result of a decline in the habitat integrity of the site, reflected in the decrease in IHAS score (Figure 4-31). This is likely due to the significant decrease in the volume of water and the lack of overhanging marginal and aquatic vegetation during the current survey as mentioned in Section 4.4.2. The decrease in ASPT score is likely a result of the significant temporal increase in EC level, and the decrease in pH level since the previous survey, which is likely to be be affecting the aquatic communities present in these systems as a result of osmotic stress. Similarly, both the SASS5 and ASPT scores have decreased at Site MMD2 KO by 7.4% and 15.5%, indicating a decline in macro-invertebrate diversity and sensitivity since April 2017. This is also likely a result of the significant temporal increase in EC level and the decrease in pH level observed at this site during the current assessment (Figure 4-32).

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Figure 4-31: Temporal Variation in SASS5, IHAS and ASPT Scores obtained during the Wet Season Surveys undertaken in April 2017 and February 2018 at Site MMD1 KAN. The Variation in EC and pH Levels are also indicated for ease of reference

Figure 4-32: Temporal Variation in SASS5, IHAS and ASPT Scores obtained during the Wet Season Surveys undertaken in April 2017 and February 2018 at Site MMD2 KO. The Variation in EC and pH Levels are also indicated for ease of reference

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4.6. Fish Community Integrity Assessment

4.6.1. Fish Habitat Assessment (FHA)

The Fish Habitat Assessment (FHA) baseline results recorded during the aquatic biomonitoring investigation for the current wet season (high flow) survey are presented in Figure 4-33. This includes data from the upstream Site MML1 on the Lilongwe River, to the Kamuzu Reservoir further downstream of the system, Site MML2 on the Lisungwi River, and Sites MML3, MML4 and MML5 on the Lilongwe River.

Figure 4-33: The Quantity and Diversity of Cover Available for the Fish Community at each Site for each Velocity-Depth Class during the Wet (High Flow) Season in February 2018

In terms of the quantity and diversity of cover available for the fish community, it is evident that slow-deep conditions dominate at Site MML1, followed by slow-shallow conditions (Figure 4-33). The fish expected at the site are likely to be limited to fish with a high intolerance for fast-flowing water and to a much lesser degree, species with a high intolerance for shallow habitats. Cover in the form of overhanging vegetation, sandy stream substrate and undercut banks is common within the slow-deep velocity-depth class, along with sufficient water depth within the water column, decreasing the risk of aerial predation. Overhanging vegetation and aquatic macrophytes are abundant within the slow-shallow velocity-depth class, providing excellent cover for fish species, although the shallow water column within this class allows for some risk of aerial predation. The presence of the dam wall downstream of this point also presents a significant migration barrier to fish thus

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limiting their movement to a large degree. At the Kamuzu Reservoir II, slow-deep conditions also dominate, with the main form of cover for fish being the deep water column. Fish species with a high intolerance for fast-flowing water and very shallow habitats are therefore likely to occur within the reservoir. The slow-shallow velocity-depth class is common along the edges of the reservoir where an abundance of overhanging vegetation and aquatic macrophytes are available for cover. The water along the edges is approximately 1 m in depth, affording more protection to fish species in relation to the shallower water at the edges of the system at Site MML1.

A greater diversity of cover is accessible to the fish community at Site MML2, with the availability of the fast-shallow velocity-depth class at this point, in addition to the slow- deep and slow-shallow classes (Figure 4-33). The fish expected in this area at the current time are likely to be limited to species with a high intolerance for very deep, fast-flowing water. Cover in the form of rocky stream substrate is available in high abundance within each of the velocity-depth classes present at this site, particularly within the fast-shallow and slow-shallow classes. Overhanging marginal vegetation is abundant within the slow- shallow and slow-deep classes, and common within the fast-shallow class, however, leafy vegetation is not available in the shallows, thus providing limited cover for fish at these points. Undercut banks and root wads are however very common within all classes which allows for good cover within this reach of the Lisungwi River. The depth of the water column within the slow-deep class also contributes to this cover. Fast-deep conditions dominate at Site MML3, followed by fast-shallow, slow-deep and slow-shallow conditions (Figure 4-33) in a few small areas, resulting from the presence of the road bridge pillars. Fish species with high intolerance values for shallow habitats and slow-flowing water are therefore likely to occur at this site. The most abundant type of cover available to fish at this site includes the water column and overhanging vegetation within the fast-deep velocity-depth class. Undercut banks are common within the fast-deep class providing some cover for fish. Rocky and sandy substrate is also considered good cover within the fast-deep class and to a lesser extent within the slow-deep, slow-shallow and fast-shallow classes as the size of the rocks appear to be smaller within these classes.

Site MML4 is dominated by slow-deep conditions within the main channel, followed by slow- shallow conditions within some areas along the banks of the system. Fish species with a high intolerance for fast-flowing water and very shallow habitats are likely to occur at this site. Abundant cover in the form of the deep water column and overhanging vegetation occurs within the slow-deep class, and to a lesser extent within the slow-shallow class. Undercut banks are common within both classes however, stream substrate cover is sparse at this site due to the lack of rocky substrate exposure. Similar conditions occur at Site MML5 in that the

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slow-deep velocity-depth class dominates at this point however, the slow-shallow class occurs over a larger area (Figure 4-33) which is due to the widening of the channel as a result of significant bank incision by livestock watering activities. As a result, the fish community expected to occur at this point are likely to have a high intolerance for very fast- flowing water. As with Site MML4, abundant cover occurs at Site MML5 in the form of overhanging vegetation and the water column, although marginal vegetation is less abundant and the water depth is shallower within the slow-shallow class due to bank erosion and channel widening. As a result, undercut banks are more abundant within this class at Site MML4. Stream substrate cover is also sparse at this site due to the lack of rocky substrate exposure.

4.6.2. The Fish Response Assessment Index (FRAI)

4.6.2.1. Reference Conditions

As previously mentioned, reference fish species lists for the Lilongwe and the Lisungwi River systems could not be derived from the FROC database or from existing literature as reference information on the frequency of occurrence of fish species within these systems is not available. Therefore, a baseline Ecological Category for fish could not be determined at this time. However, the data obtained during this wet season survey should be utilised as reference values in the FRAI index for Ecological Category determination during future surveys. Of these fish species, five are included in the FRAI index (Kleynhans, 2007). For those not included in the index, species of the same genus, family or class with similar preferences, tolerances and environmental requirements were used to represent these species in the index. The reference fish community, along with the associated FRAI abbreviation code, relative abundance, determined FROC and FROC confidence value for each species is presented in Table 4-9.

The reference species list includes 15 indigenous fish species for the Lilongwe River system and 5 indigenous species for the Lisungwi River system. Reference species within the Lilongwe system include Astatotilapia calliptera (Eastern River Bream), Chiloglanis neumanni (Neumann’s Rock Catlet), Clarias gariepinus (Sharptooth Catfish), various Enteromius species, including E. choloensis (Silver Barb), E. kerstenii (Redspot Barb), E. macrotaenia (Broadband Barb), E. paludinosus (Straightfin Barb), E. toppini (East Coast Barb) and E. trimaculatus (Threespot Barb); Labeobarbus johnstonii (Johnstonii Yellowfish), Mastacembelus shiranus (Malawi Spinyeel), Opsaridium tweddleorum (Dwarf Sanjika), Oreochromis karongae (Karonga tilapia), Oreochromis lidole (Lidole tilapia) and Pseudocrenilabrus philander (Southern Mouthbrooder). Reference species within the Lisungwi River system include Chiloglanis neumanni, Clarias gariepinus, Labeobarbus johnstonii, Mastacembelus shiranus and Oreochromis lidole.

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Table 4-9: Reference Fish Species List derived during the February 2018 Wet (High Flow) Season for the Lilongwe and Lisungwi Rivers

Species FRAI Code Relative Abundance Determined FROC Confidence

Lilongwe River (Including the Kamuzu Reservoir)

Astatotilapia calliptera OPLA 2 4 5

Chiloglanis neumanni CPAR 1 1 5

Clarias gariepinus CGAR 1 2 4

Enteromius choloensis BEUT 1 2 4

Enteromius kerstenii BBRI 1 1 4

Enteromius macrotaenia BRAD 2 2 4

Enteromius paludinosus BPAU 2 2 4

Enteromius toppini BTOP 2 2 4

Enteromius trimaculatus BTRI 2 2 4

Labeobarbus johnstonii BNAT 1 1 4

Mastacembelus shiranus AMOS 1 3 4

Opsaridium tweddleorum OPER 2 3 4

Oreochromis karongae ONIL 2 2 4

Oreochromis lidole OMOS 3 5 4

Pseudocrenilabrus PPHI 1 1 3 philander

Lisungwi River

Chiloglanis neumanni CPAR 2 1 3

Clarias gariepinus CGAR 1 1 3

Labeobarbus johnstonii BNAT 1 1 3

Mastacembelus shiranus AMOS 2 1 3

Oreochromis lidole OMOS 1 1 3

*Abundance: 1 = 1 to 5 fish / 2 = 6 to 50 fish / 3 = >50 fish *FROC: 1 = 20% / 2 = 40% / 3 = 60% / 4 = 80% / 5 = 100% *Confidence: 1 = Low / 2 = Low to Moderate / 3 = Moderate / 4 = Moderate to High / 5 = High

A. calliptera occurs in a wide variety of riverine and marshy habitats (Skelton, 2001). The Chiloglanis sp. show high preferences for fast-flowing water with rocky substrate and C.

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gariepinus is completely omnivorous and has a high preference for slow-flowing water. Similarly, the Enteromius species prefer slow flows and have a high preference for overhanging vegetation (Kleynhans, 2007), whereas L. johnstonii has a preference for moderate to fast-flowing water (Kadye, 2008). M. shiranus and O. tweddleorum have a high affinity for shallow, rocky and sandy substrate (Konings, 1990, Skelton, 2001). O. karongae and O. lidole were sampled in slow-shallow and slow-deep habitats indicating an affinity for slow-flowing water. P. philander has a preference for slow and shallow habitats, overhanging vegetation and undercut banks (Kleynhans, 2007).

Of these fish species, five are included in the FRAI index, namely, Clarias gariepinus, Enteromius paludinosus, Enteromius toppini, Enteromius trimaculatus and Pseudocrenilabrus philander (Kleynhans, 2007). For those not included in the index, species of the same genus with similar preferences, tolerances and environmental requirements were used to represent these species in the index, with the exception of Astatotilapia calliptera as fish of the same genera are not available in the index. In this case, fish from the same family with similar preferences and tolerances was used to represent this species in the index. Therefore, Astatotilapia calliptera is represented by Oreochromis placidus (OPLA). In the case of the Longtail Spiny Eel, Mastacembelus shiranus, however, a representative of the same family is not available as the only family of eels that occur within the FRAI index are the Freshwater Eels, Anguillidae. These eels are of the same Class, Actinopterygii, commonly known as the Ray-Finned fishes, but are of a different order, namely the Anguilliformes, or Eels and Morays. M. shiranus belongs to the Order Synbranchiformes, or Spiny-Eels. As both eels are tropical, freshwater species with a tolerance for brackish water (Skelton, 2001), similar preferences exist between the two. M. shiranus is therefore represented by Anguilla mossambica (AMOS), the Longfin Eel, in the index as this species also has a preference for flowing water within shallow, rocky substrate (Kleynhans, 2007).

4.7. Mollusc Assessment

Lake Malawi is one of the major centres of endemism with regards to freshwater molluscs in Africa (Day and De Moor, 2002). Within Lake Malawi, there are 27 species of freshwater gastropod fauna (univalves), 17 of which are endemic to the area (Brown, 1994; Seddon et al., 2011). Of these 17 species, 15 are prosobranchs and 2 are pulmonates. With regards to freshwater bivalve fauna, 8 species occur within Lake Malawi, 3 of which are endemic (Seddon et al., 2011). Reference species lists for molluscs within the Lilongwe River system, including the Kamuzu Reservoir, and the Lisungwi River, also could not be derived from existing literature as reference information is not available. Furthermore, no species were

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available for sampling at Site MML2 on the Lisungwi River. Therefore, the reference mollusc species list for the Lilongwe River reach and the two dambos is presented in Table 4-10, along with the respective common names or synonyms for each species (if applicable), the relative abundance, the determined FROC and the FROC confidence value.

Table 4-10: Reference Mollusc Species List derived during the February 2018 Wet (High flow) Season for the Lilongwe River Reach and the Two Dambo Systems

Common Relative Species Determined FROC Confidence Name / Synonym Abundance

Lilongwe River (Including the Kamuzu Reservoir)

Biomphalaria pfeifferi Bloodfluke Planorb 2 2 2

Bulinus globosus Physa globosus 2 4 3

Aspatharia Chambardia wahlbergi 1 1 2 wahlbergi

Coelatura N/A 1 2 3 mossambicensis

Gyraulus connollyi N/A 1 1 2

Lanistes ellipticus N/A 1 2 2

Lymnaea natalensis Radix natalensis 2 3 3

Dambos

Bulinus globosus Physa globosus 2 3 3

*Abundance: 1 = 1 to 5 molluscs / 2 = 6 to 50 molluscs / 3 = >50 molluscs *FROC: 1 = 20% / 2 = 40% / 3 = 60% / 4 = 80% / 5 = 100% *Confidence: 1 = Low / 2 = Low to Moderate / 3 = Moderate / 4 = Moderate to High / 5 = High

4.8. Metal Analysis

The results of the baseline metal analysis conducted on whole fish tissue samples of Astatotilapia calliptera from Sites MML1, MML2, MML3, MML4 and the Kamuzu Reservoir II, and Oreochromis lidole from Sites MML1, MML4 and the Kamuzu Reservoir II are indicated in Table 4-11 and discussed below. An indication of baseline metal concentrations within the native bivalve mollusc, Coelatura mossambicensis, sampled from Site MML4 on the Lilongwe River system and sediment from the Kamuzu Reservoir II is also provided in Table 4-12 and Table 4-13, respectively. Refer to Appendix A for the full analytical reports.

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Table 4-11: Baseline Metal Analysis Results of Whole Fish Tissue Sampled from the Lilongwe River, the Lisungwi River and the Kamuzu Reservoir II during the Wet (High Flow) Season in February 2018

Concentration (mg/kg) wet weight Detection Limit Guideline Kamuzu Metal MML1 MML2 MML3 MML4 (mg/l) (mg/kg) wet weight Reservoir II

A O A A A O A O

Aluminium as Al <0.100 - 1716.7 7175.2 45.98 229.64 301.53 1666.29 33.83 74.86

Arsenic as Ar <0.010 1.5 (CEPA, 2000; FAO, 0.29 0.81 0.05 0.14 0.28 0.24 0.04 0.07 1983) 0.05 (EU, 2006) and Cadmium as Cd <0.010 0.3 (CEPA, 2000; FAO, 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1983)

Chromium as Cr (Total) <0.010 1.0 (CEPA, 2000; FAO, 1.66 4.18 0.25 0.63 0.31 11.37 0.04 0.02 1983)

Copper as Cu <0.010 20 (CEPA, 2000; FAO, 2.08 9.58 1.09 0.89 2.36 1.63 0.61 1.62 1983)

Iron as Fe <0.025 - 1627.6 6864.8 100.43 259.37 373.87 1458.1 76.05 127.69

Dissolved Mercury as Hg <0.010 0.5 (EU, 2006; CEPA, 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2000; FAO, 1983)

Manganese as Mn <0.025 - 59.19 186.1 29.63 98.19 407.82 66.90 17.02 37.32 0.3 (EU, 2006) and Lead as Pb <0.010 2 (CEPA, 2000; FAO, 0.28 1.29 0.04 0.04 0.06 0.26 0.0 0.14 1983)

Selenium as Se <0.010 2 (CEPA, 2000; FAO, 0.62 0.34 0.65 0.31 0.09 0.82 0.37 0.36 1983) *A = Astatotilapia calliptera / *O = Oreochromis lidole *Values in red indicate guideline exceedance.

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The results in Table 4-11 indicate that the lead (Pb) concentration measured in O. lidole from Site MML1 exceeds the Quality Guideline Levels for Human Consumption specified by EU (2006). According to the Median International Standards for Trace Elements (CEPA, 2000; FAO, 1983) the chromium (Cr) concentration guideline is exceeded in both A. calliptera and O. lidole from Site MML1 and in O. lidole from Site MML4. This suggests that some impact from existing sources or activities may be occurring at these points during the 2018 wet season survey. Although there are currently no standards available for aluminium (Al), iron (Fe) and manganese (Mn) in fish tissue, the concentrations obtained during future surveys may be compared to the concentrations obtained during the current survey in order to identify and monitor any increases over time.

Table 4-12: Baseline Metal Analysis Results of the Native Bivalve Mollusc, Coelatura mossambicensus, sampled from Site MML4 on the Lilongwe River during the Wet (High Flow) Season in February 2018

Detection Limit Guideline Concentration Metal (mg/l) (mg/kg) wet weight (mg/kg) wet weight

Aluminium as Al <0.100 - 53.34

1.4 Arsenic as Ar <0.010 0.03 (CEPA, 2000; FAO, 1983) 1 Cadmium as Cd <0.010 (EU, 2006; CEPA, 2000; FAO, 0.0 1983) 1 Chromium as Cr (Total) <0.010 0.06 (CEPA, 2000; FAO, 1983) 20 Copper as Cu <0.010 0.63 (CEPA, 2000; FAO, 1983)

Iron as Fe <0.025 - 58.03

0.5 Dissolved Mercury as Hg <0.010 0.0 (CEPA, 2000; FAO, 1983)

Manganese as Mn <0.025 - 6.10

1.5 (EU, 2006) and Lead as Pb <0.010 0.02 2 (CEPA, 2000; FAO, 1983) 0.3 Selenium as Se <0.010 0.16 (CEPA, 2000; FAO, 1983) *Values in red indicate guideline exceedance.

With regards to the metal analysis results for the bivalve mollusc, C. mossambicensus, the results presented in Table 4-12 indicate that none of the guideline concentration values presented in the Median International Standards for Trace Elements (CEPA, 2000; FAO, 1983) and the Quality Guideline Levels for Human Consumption (EU, 2006) are exceeded within the

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mussel tissue. Similarly to the metal guideline concentration values for fish tissue, there are currently no standards available for aluminium (Al), iron (Fe) and manganese (Mn) in bivalve tissue. However, concentrations obtained during future surveys may also be compared to the concentrations obtained during the current survey in order to identify and monitor any increases over time.

Table 4-13: Metal Analysis Results in Sediment Sampled from the Kamuzu Reservoir II during the Wet (High Flow) Season in February 2018

Detection Guideline Concentration Metal Limit (mg/l) (mg/kg) dry weight (mg/kg) dry weight

Aluminium as Al <0.100 - 53294.8

9.79 (US EPA, 2006; MacDonald et al., Arsenic as Ar <0.010 2000) and 1.60 5.9 (CCME, 2001) 0.99 (US EPA, 2006; MacDonald et al., Cadmium as Cd <0.010 2000) and 0.0 0.6 (CCME, 2001) 43.4 (US EPA, 2006; MacDonald et al., Chromium as Cr (Total) <0.010 2000) and 328.4 37.3 (CCME, 2001) 31.6 (US EPA, 2006; MacDonald et al., Copper as Cu <0.010 2000) and 79.5 35.7 (CCME, 2001) 20 000 (US EPA, Iron as Fe <0.025 73250.5 2006) 0.18 (US EPA, 2006; MacDonald et al., Dissolved Mercury as Hg <0.010 2000) and 0.0 0.13 (CCME, 2001)

Manganese as Mn <0.025 460 (US EPA, 2006) 1234.9

35.8 (US EPA, 2006; MacDonald et al., Lead as Pb <0.010 2000) and 5.99 35 (CCME, 2001)

Selenium as Se <0.010 2 (US EPA, 2006) 0.0

*Values in red indicate guideline exceedance.

From the results indicated in Table 4-13, the chromium (Cr) concentrations within the sediment sampled from the Kamuzu Reservoir II exceed the Threshold Effect Concentration (TEC) specified by MacDonald et al. (2000) and Persaud et al. (1993: In US EPA, 2006) (TEC:

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43.4 mg/kg) and the Interim Sediment Quality Guidelines (ISQG) specified by CCME (2001) (TEC: 37.3 mg/kg). This suggests that an impact may be occurring at this point during the current survey. Similarly, the copper (Cu) concentration exceeds the screening guideline specified by MacDonald et al. (2000) and Persaud et al. (1993: In US EPA, 2006) (TEC: 31.6 mg/kg), and the ISQG specified by CCME (2001) (TEC: 35.7 mg/kg). The iron (Fe) and manganese (Mn) concentrations also exceeds the screening guideline specified by Persaud et al. (1993) (In US EPA, 2006) (TEC: 20 000 mg/kg for Fe and 460 mg/kg for Mn). There are currently no sediment quality guidelines available for aluminium (Al), however, concentrations obtained during future surveys may be compared temporally to the data obtained during the current survey in order to identify and monitor any increases over time.

4.9. Diatom Analysis

A total of 65 diatom species were recorded at the eight sites and the dominant diatom species recorded at all sites included Nitzschia sp. and Gomphonema parvulum (Table 4-14) (Ecotone, June 2018). Based on the diatom community analyses, the water quality at each site appeared to have some pollution-related impacts and the overall water quality was Moderate for all sites. (Van Dam et al., 1994; Taylor et al., 2007b; Cantonati et al., 2017; Table 4-14 and Table 4-15). It is important to know that the dominant species mentioned above are cosmopolitan and have very wide ecological amplitudes. Hence, the predominance of these species at specific sites should be analysed with caution. It is also important to consider the diatom assemblage as a whole, with the dominant species being the main focus. According to temporal diatom analysis trends, the ecological water quality has shown a general improvement from the April 2017 to the February 2018 surveys and a stable trend between the previous October 2017 and the current February 2018 survey (Table 4-15 to Table 4-18). Additional information is provided below for the sub-dominant species in order to make ecological inferences for the eight sites assessed (Taylor et al., 2007b; Cantonati et al., 2017). Refer to Appendix B for the full analytical report.

4.9.1. Site MML1

During the current survey, this site is dominated by Nitzschia sp. which points to eutrophic, polluted (α-mesosaprobic to polysaprobic) freshwater. The dominance of Navicula sp. suggests alkaline, low temperature, eutrophic running water with medium-high conductivity and species from this genus are commonly found in organically polluted water. The presence of G. parvulum indicates impacts associated with agricultural run-off and is adapted to withstand physical disturbance and benefits from organic enrichment. The presence of Orthoseira roeseana points to aerial habitats indicating that this site may experience

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periodic drying out. The Gomphonema sp. can be found in clean waters as well as water receiving sewage effluent. The diatom assemblage at this site indicates alkaline, eutrophic conditions with electrolyte-rich content (Table 4-14). Owing to the presence of the dominant species, this site appears to be impacted by organic/anthropogenic pollution which is possibly associated with the surrounding land-use. The %PTV score is relatively low indicating that there is a serious impact associated with organic pollution at the time of the survey (Table 4-15). A slight decrease in the level of organic pollution is evident since the previous survey (Figure 4-34) and the overall water quality appears to remain stable and is considered Moderate (Table 4-15 and Table 4-18).

4.9.2. Site MML2

The overwhelming dominance of G. parvulum and G. rhombicum indicates oligosaprobic and mesosaprobic, oligo- to eutrophic, electrolyte-poor freshwater and points to impacts associated with agricultural run-off. The subdominance of Nitzschia sp. points to α- mesosaprobic to polysaprobic freshwater and this taxon is commonly found in untreated waste water and in habitats that are strongly impacted by industrial sewerage. The subdominant, N. symmetrica and Navicula sp. points to eutrophic, electrolyte-rich waters and both taxa are tolerant of strongly polluted conditions. The presence of Achananthidum sp. has been recorded in high proportions over a wide range of trophic levels and is usually absent from moderately- to strongly-acidic or very electrolyte-poor environments. The diatom community at this site points to meso- to eutrophic conditions with moderate electrolyte content and owing to the presence of the dominant species there appears to be some form of organic pollution impacts at this site (Table 4-14). The %PTV score is relatively high indicating that there is some impact associated with organic pollution, possibly originating from the surrounding land-use (Table 4-15). The level of organic pollution shows a slight increase since the previous survey (Figure 4-34) however, the overall water quality is considered Moderate (Table 4-15).

4.9.3. Site MML3

The dominance of Achnanthidium sp. indicates clean and polluted waters, including those affected by acid mine drainage (Ponader & Potapova, 2007) and is usually absent from moderately- to strongly-acid or very electrolyte poor environments. The subdominance of Nitzschia sp. and N. amphibia points to eutrophic freshwater and are tolerant to strongly polluted conditions. Nitzschia sp., in particular, are commonly found in untreated waste water and in habitats that are strongly impacted by industrial sewerage. The presence of Amphora veneta indicates elevated electrolyte content, tolerating heavy pollution. The

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presence of G. parvulum indicates oligosaprobic and mesosaprobic, oligo- to eutrophic freshwater and points to impacts associated with agricultural run-off. The presence of Cyclotella meneghiniana points to eutrophic, electrolyte rich rivers (Table 4-14). The diatom assemblage at this site indicates eutrophic, moderate electrolyte freshwater conditions. The %PTV score is relatively low but owing to the presence of Nitzschia sp., this site appears to be slightly impacted by some form of organic/anthropogenic pollution entering the system. The level of organic pollution decreased since the previous survey (Figure 4-34), but the overall water quality has remained stable and is considered Moderate (Table 4-15 and Table 4-18).

4.9.4. Site MML4

This site is dominated by Achnanthidium sp. which points to calcium-bicarbonate rich, mesotrophic to eutrophic freshwater with medium-high conductivity. The subdominance of Nitzschia sp. and N. amphibia points to eutrophic freshwater and are tolerant to strongly polluted conditions. The subdominance of Cyclotella meneghiniana points to eutrophic, electrolyte rich rivers. The presence of Navicula sp. suggests alkaline, low temperature, eutrophic running water with medium-high conductivity and species from this genus are commonly found in organically polluted water. The presence of Ulnaria ulna indicates alkaline, medium conductivity, oligosaprobic, moderately eutrophic habitat (Table 4-14). The diatom community at this site points to meso- to eutrophic conditions with moderate electrolyte content. Owing to the presence of Achnanthidium sp. and the relatively low %PTV score, this site appears to have a low impact associated with organic pollution. The overall water quality is considered Moderate (Table 4-15).

4.9.5. Site MML5

This site is dominated by Navicula sp. which suggests alkaline, low temperature, eutrophic running water with medium-high conductivity and species from this genus are commonly found in organically polluted water. The subdominance of Cyclotella meneghiniana points to eutrophic, electrolyte rich rivers. The subdominance of Orthoseira roeseana and Navicula erifuga points to brackish conditions and both species are adapted to intermittently wet conditions. The presence of Nitzschia sp. points to eutrophic, polluted (α-mesosaprobic to polysaprobic) freshwater (Table 4-14). The diatom community at this site points to slightly brackish, eutrophic conditions with electrolyte-rich content. The %PTV score is low indicating that there is no serious impact associated with organic pollution and the overall water quality is considered Moderate (Table 4-15).

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4.9.6. Site MMDR

This site is dominated by Nitzschia sp. and N. sigma which points to eutrophic, electrolyte- rich and brackish conditions. The subdominance of Pinnularia gibba points to calcium- bicarbonate-poor freshwater and may occur in rivers strongly impacted by waste water. The presence of Gyrosigma acuminatum indicates alkaline freshwater with high trophic levels and sensitive to strongly polluted conditions. The presence of Frustulia crassinervia points to dystrophic, electrolyte-poor freshwater and this taxon is usually an indicator of good ecological quality. The presence P. subcapitata points to oligotrophic, electrolyte-poor and weakly acidic conditions. The occurrence of Navicula sp. suggests alkaline, low temperature, eutrophic running water with medium-high conductivity and species from this genus are commonly found in organically polluted water (Table 4-14). The diatom community at this site points to slightly brackish, meso- to eutrophic conditions with moderate electrolyte content. The %PTV score is relatively low indicating that there is no serious impact associated with organic pollution. The overall ecological water quality indicates a stable trend since the previous survey and is considered Moderate (Table 4-15 and Table 4-18).

4.9.7. Site MMD1 KAN

The overwhelming dominance of Nitzschia sp. points to eutrophic, polluted (α-mesosaprobic to polysaprobic) freshwater. The subdominance of Pinnularia marchica points to alkaline, strongly eutrophic, moderately polluted running waters with medium electrolyte content, particularly developed under conditions of cold water temperature. The presence of Pinnularia sp. points to calcium-bicarbonate-poor freshwater and may occur in rivers strongly impacted by waste water. The presence of Stauroneis anceps points to oligo- to eutrophic, but oligosaprobic freshwater with medium electrolyte content. The presence of P. borealis indicates pseudaerial habitats, such as soils, bryophyte carpets and rock walls (Table 4-14). The diatom community at this site points to alkaline, meso- to eutrophic conditions with moderate electrolyte content and owing to the presence of Nitzschia, there appeared to be some form of pollution impact at this site. The %PTV score indicates that there is no evidence of organic pollution suggesting that some other form of pollution is impacting this site. The overall water quality is considered Moderate and has improved since the previous survey (Table 4-15 and Table 4-18).

4.9.8. Site MMD2 KO

The overwhelming dominance of Nitzschia sp. points to eutrophic, polluted (α-mesosaprobic to polysaprobic) freshwater. The subdominance of G. parvulum and Gomphonema sp. indicated oligosaprobic and mesosaprobic, oligo- to eutrophic, electrolyte-poor freshwater and points to impacts associated with agricultural run-off. The subdominance of Adlafia

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bryophila points to a broad electrolyte content range and this taxon is sensitive to organic pollution. The presence of Pinnularia sp. points to calcium-bicarbonate-poor freshwater and may occur in rivers impacted by waste water. The presence of Sellaphora pupula indicates alkaline, eutrophic to polytrophic conditions with medium to increased electrolyte content (Table 4-14). The diatom community at this site points to alkaline, eutrophic conditions with moderate electrolyte content. Owing to the dominance of Nitzschia sp. and the relatively high %PTV score, this site appears to be impacted by some form of organic/anthropogenic pollution. The overall water quality is considered Moderate (Table 4-15).

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Table 4-14: Species List for each Site indicating Abundance and Dominance of Diatom Species in February 2018

MMD1 MMD2 Taxa MML1 MML2 MML3 MML4 MML5 MMDR KAN KO Achnanthidium sp. 40 78 64 7 5 10 Adlafia bryophila 7 5 4 36 (Petersen) Moser Lange- Bertalot & Metzeltin Amphora veneta 14 13 30 10 3 4

Aulacoseira granulata 16 20 28 2

Caloneis fontinalis (Grun.) 4 Lange-Bertalot & Reichardt Caloneis lancettula 4 4 (Schulz) Lange-Bertalot & Witkowski Cocconeis placentula 6 8 2 Ehrenberg var. placentula Craticula ambigua 6 (Ehrenberg) Mann Craticula buderi (Hustedt) 0 8 11 Lange-Bertalot Craticula molestiformis 20 7 15 (Hustedt) Lange-Bertalot Craticula minusculoides 10 18 6 16 (Hustedt) Lange-Bertalot Cyclotella meneghiniana 18 16 30 56 4 4 Kützing Cymbella tumida 6 6 3 (Brebisson)Van Heurck Cymbella turgidula 2 3 Grunow 1875 in A.Schmidt & al. var. turgidula Diadesmis confervacea 20 Kützing var. confervacea Encyonema minutum 12 10 4 10 10 Encyonema silesiacum 14 4 12 (Bleisch in Rabh.) D.G. Mann 4 Epithemia sorex Kützing Eunotia botuliformis Wild 4 4 Norpel & Lange-Bertalot Eunotia minor (Kützing) 4 Grunow in Van Heurck Eunotia sp. 4

Fragilariforma virescens 14

Fragilaria dilatata 22 (Brébisson) Lange-Bertalot Fragilaria species 8 42 15 38 4 4

Frustulia asiatica 4 5 (skvortzow) Metzeltin

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MMD1 MMD2 Taxa MML1 MML2 MML3 MML4 MML5 MMDR KAN KO Lange-Bertalot & Nergui

Frustulia crassinervia 7 7 3 36 (Breb.) Lange-Bertalot et Krammer Gomphonema 20 4 minutum(Ag.)Agardh f. minutum Gomphonema parvulum 26 110 15 16 6 78

Gomphonema rhombicum 35 18 M. Schmidt Gomphonema species 30 12 25 20 12 6 4 25

Gyrosigma acuminatum 10 5 5 3 8 34 5 (Kützing)Rabenhorst Hippodonta capitata 4 6

Lemnicola hungarica 4 (Grunow) Round & Basson Luticola species 4

Navicula erifuga Lange- 12 17 28 Bertalot Navicula radiosa 12 17 28 3

Navicula rostellata 5 10

Navicula sp. 86 22 7 28 68 5 4 12 Navicula symmetrica 39 12 19 Patrick Neidium 4 15 4 affine(Ehrenberg)Pfitzer Neidium bisulcatum 4 (Lagerstedt) Cleve Nitzschia amphibia 10 40 23 10 10

Nitzschia sigma 9 28 7 8 38 (Kützing)W.M.Smith Nitzschia sp.1 88 35 48 50 50 48 168 108

Orthoseira roeseana 32 4 20 36 5 (Rabenhorst) O'Meara Pinnularia obscura Krasske 4

Pinnularia rhombarea 12 0 Krammer var.halophila Krammer Pinnularia borealis 4 30 10 Ehrenberg var. borealis Pinnularia gibba 7 42 42 4 Ehrenberg Pinnularia marchica Ilka 0 94 Schönfelder var. marchica Pinnularia obscura Krasske 25

Pinnularia sardoelliptica 13 32 Lange-Bertalot & Metzeltin Pinnularia subcapitata 24 Gregory var. subcapitata 12 12 8 Placoneis symmetrica

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MMD1 MMD2 Taxa MML1 MML2 MML3 MML4 MML5 MMDR KAN KO (Hustedt) Lange-Bertalot Placoneis undulata 4

Planothidium 4 frequentissimum(Lange- Bertalot)Lange-Bertalot Sellaphora pseudopupula 4 (Krasske) Lange-Bertalot Sellaphora pupula 4 4 2 22 (Kützing) Mereschkowksy Sellaphora species 11

Stauroneis anceps 6 28 Ehrenberg Surirella minuta Brebisson 4 4

Surirella sp.1 6

Surirella terricola Lange- 14 4 6 Bertalot & Alles Tabularia fasciculata 6 (Agardh)Williams et Round Ulnaria ulna (Nitzsch.) 18 7 10 3 4 Compère Total 450 450 450 450 450 450 450 450 Nutrients Salinity Organics pH-Alkaline pH-Slightly Acidic Other Dominant

Table 4-15: Diatom Index Scores for the Study Sites indicating the Ecological Water Quality obtained in February 2018

Site %PTV SPI BDI Quality

MML1 10.2 9.8 12 Moderate MML2 26.4 10.9 11.6 Moderate MML3 9.6 11 15.4 Moderate MML4 5.1 11 10.5 Moderate MML5 3.1 11.8 14.2 Moderate MMDR 10.9 12.3 11.6 Moderate MMD1 KAN 0 9.8 13.6 Moderate MMD2 KO 17.3 9.7 11.2 Moderate

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Table 4-16: Diatom Index Scores for the Study Sites indicating the Ecological Water Quality in October 2017 (Walsh, November 2017)

Site %PTV SPI BDI Quality

MML1 13.7 12.5 12.2 Moderate MML2 21.4 10.8 14.3 Moderate MML3 20.3 10.9 12.4 Moderate MMDR 17.7 11.8 12.8 Moderate MMD1 KAN 32 8 9.4 Poor

Table 4-17: Diatom Index Scores for the Study Sites indicating the Ecological Water Quality in April 2017 (Walsh, June 2017)

Site %PTV SPI BDI Quality

MML1 41.5 4.22 3.38 Moderate MML2 26.3 4.01 3.25 Moderate MML3 8.3 4.58 3.49 Moderate MMDR 53.5 4.54 3.07 Moderate MMD1 KAN 48.3 4.19 2.75 Poor

Figure 4-34: Temporal Variation in %PTV for the Monitoring Points on the Aquatic Resources in the vicinity of the Project Area from April 2017 to February 2018

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According to the temporal diatom trend analysis, the ecological water quality has shown a general improvement from the April 2017 to the February 2018 surveys (Table 4-18). In comparison to the previous October 2017 survey, Site MMD1 KAN showed an improvement in water quality from Poor to Moderate. This slight improvement is likely associated with the decrease in organic pollution at this site (Table 4-15; Table 4-16; Table 4-18; Figure 4-34). The water quality at the remainder of the sites showed a stable trend (Table 4-18), despite the slight decrease in organic pollution from October 2017 to February 2018 at Sites MML1, MML3, MMDR and MMD1 KAN. Site MML2 showed a slight increase in organic pollution within this time period but the level was low indicating that there were no serious impacts at this site (Figure 4-34).

Table 4-18: Diatom Index Scores for the Study Sites indicating the Ecological Water Quality Trends between Seasons

Quality Quality Quality Site Trend (April 2017) (October 2017) (February 2018) MML1 Bad Moderate Moderate ► MML2 Bad Moderate Moderate ► MML3 Bad Moderate Moderate ► MML4 - - Moderate - MML5 - - Moderate - MMDR Bad Moderate Moderate ► MMD1 KAN Bad Poor Moderate ▲ MMD2 KO Insufficient Cells Dry Moderate -

4.10. Whole Effluent Toxicity (WET) Testing

During the wet season (high flow) aquatic assessment, toxicological testing was conducted on samples obtained from eight biomonitoring points in February 2018. The results are presented and discussed in the section to follow. Refer to Appendix C for the full analytical report. The toxicological testing results of the water at each monitoring point obtained during the wet season survey are summarised in Table 4-19.

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Table 4-19: Summary of the Results obtained for Toxicological Testing in February 2018

Selenastrum Vibrio fischeri Daphnia Poecilia Sampling capricornutum (% inhibition/ Magna reticulata Hazard Class Point (% stimulation) (% mortality) (% mortality) inhibition/stimulation) MML1 -12 5 -5 -8 Class I

MML2 -9 12 0 0 Class I

MML3 -9 13 0 0 Class I

MML4 -13 18 0 -8 Class I

MML5 -18 10 0 0 Class I

MMDR -16 14 -10 0 Class I MMD1 -32 5 0 0 Class II KAN MMD2 KO -26 ** -5 0 Class II Kamuzu Reservoir -18 17 0 0 Class I II ** Algal test result inconclusive due to interference caused by precipitate forming in the sample during testing. As the degree of inhibition/stimulation is unknown, individual test result was not used for overall hazard classification.

Results of the screening tests conducted on the water samples from each site indicate that the water poses no acute hazard (Class I) to the aquatic communities at Sites MML1 to MML5 as well as at Site MMDR and the Kamuzu Reservoir II during the current assessment. The water at Sites MMD1 KAN and MMD2 KO however, were found to pose a slight acute hazard (Class II).

The levels of bacterial growth inhibition are low at all sites, with the exception of Sites MMD1 KAN and MMD2 KO, where bacterial growth inhibition is above 20%. The highest level of bacterial inhibition occurs at Site MMD1 KAN. Low levels of algal stimulation are evident at all sites. Low mortality rates of D. magna, which represents the macro-invertebrate community, are evident at Sites MML1, MMDR and MMD2 KO. Low mortality rates of P. retuculata, which represents the fish community, have also been recorded at Sites MML1 and MML4. This is supported by the metal analysis results that indicate a guideline exceedance in the lead (Pb) concentration measured in O. lidole from Site MML1 and the chromium (Cr) concentration measured in both A. calliptera and O. lidole from Site MML1 and in O. lidole from Site MML4.

Since the previous wet season assessment in April 2017, the toxicological hazard has improved from a slight acute hazard (Class II) condition to a no acute hazard (Class I) condition at Site MML1, from an acute hazard (Class III) condition to a no acute hazard (Class

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I) condition at Sites MML2 and MML3 and from a high acute hazard (Class IV) condition to a no acute hazard (Class I) condition at Site MMDR during the current assessment. The toxicological hazard has remained in a slight acute hazard (Class II) condition at Sites MMD1 KAN and MMD2 KO.

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5. CONCLUSION

With regards to the fish and mollusc baseline survey, results of the Fish Habitat Assessment (FHA) indicate that slow-deep conditions dominate at Site MML1, followed by slow-shallow conditions. The fish expected at the site are likely to be limited to fish with a high intolerance for fast-flowing water and shallow habitats. In contrast, fast-shallow, slow- deep and slow-shallow classes occur at Site MML2. The fish expected in this area at the current time are likely to be limited to species with a high intolerance for very deep, fast- flowing water. Fast-deep conditions dominate at Site MML3, followed by fast-shallow, slow- deep and slow-shallow conditions in a few small areas. Fish species with a high intolerance for shallow habitats and slow-flowing water are therefore likely to occur at this site. Site MML4 is dominated by slow-deep conditions within the main channel, followed by slow- shallow conditions within some areas along the banks of the system. Fish species with a high intolerance for fast-flowing water and very shallow habitats are likely to occur at this site. Slow-deep conditions dominate at Site MML5 however, the slow-shallow class occurs over a larger area. As a result, the fish community expected to occur at this point are likely to have a high intolerance for very fast-flowing water.

Reference fish species lists for the Lilongwe River, including the Kamuzu Reservoir (as it is on the same system), and the Lisungwi River system, could not be derived from the FROC database or from existing literature as reference information on the frequency of occurrence of fish species within these systems is not available. Therefore, a baseline Ecological Category for fish could not be determined at this time. However, the data obtained during this wet season survey should be utilised as reference values in the FRAI index for Ecological Category determination during future surveys. The reference species list includes 15 indigenous fish species for the Lilongwe River system and 5 indigenous species for the Lisungwi River system. Reference species within the Lilongwe system include Astatotilapia calliptera (Eastern River Bream), Chiloglanis neumanni (Neumann’s Rock Catlet), Clarias gariepinus (Sharptooth Catfish), various Enteromius species including E. choloensis (Silver Barb), E. kerstenii (Redspot Barb), E. macrotaenia (Broadband Barb), E. paludinosus (Straightfin Barb), E. toppini (East Coast Barb) and E. trimaculatus (Threespot Barb); Labeobarbus johnstonii (Johnstonii Yellowfish), Mastacembelus shiranus (Malawi Spinyeel), Opsaridium tweddleorum (Dwarf Sanjika), Oreochromis karongae (Karonga tilapia), Oreochromis lidole (Lidole) and Pseudocrenilabrus philander (Southern Mouthbrooder). Reference species within the Lisungwi River system include Chiloglanis neumanni, Clarias gariepinus, Labeobarbus johnstonii, Mastacembelus shiranus and Oreochromis lidole.

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Reference species of mollusc within the Lilongwe system include Biomphalaria pfeifferi, Bulinus globosus, Chambardia wahlbergi, Coelatura mossambicensis, Gyraulus connollyi, Lanistes ellipticus and Lymnaea natalensis. Bulinus globosus was the only species present within the dambos during the current survey.

Regarding the biomonitoring assessment, the in situ water quality at Sites MML1, MML2, MML4 and MML5 during the wet (high flow) season biomonitoring assessment undertaken in February 2018 are considered to be fair. It is evident that the EC levels at Sites MML1 and MML2 have increased since the previous wet season survey undertaken in April 2017. As the systems were in flood during the previous survey, it is likely that the nutrients were diluted resulting in the low EC values observed in April 2017. During the survey conducted in February 2018 however, these systems were not in flood, resulting in less dilution and higher readings. Poor water quality conditions are evident at Site MML3, with a significant increase in EC level and an increase in the pH level; and at Sites MMDR, MMD1 KAN and MMD2 KO with an increase in EC levels (significant increases in EC levels have occurred at the two dambo sites) and a decrease in pH and DO levels occurring at these sites in relation to the previous survey.

The results of the IHIA assessment indicate that the general habitat integrity may be regarded as being Moderately Modified (Class C) at Sites MML1, MML2 and MMD1 KAN, Largely Modified (Class D) at Sites MML3 and MML5 and Extensively Modified (Class E) at Sites MML4, MMDR and MMD2 KO. Since the previous wet season survey conducted in April 2017, the general habitat integrity has remained in a Moderately Modified (Class C) condition at Sites MML1 and MML2, has declined from a Largely Natural with Few Modifications (Class B) condition to a Largely Modified (Class D) condition at Site MML3, has declined from an Unmodified, Natural (Class A) condition to a Moderately Modified (Class C)

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condition at Site MMD1 KAN and from a Largely Modified (Class D) condition to an Extensively Modified (Class E) condition at Site MMD2 KO. The habitat integrity at Site MMDR has however improved from a Critically Modified (Class F) condition in April 2017 to an Extensively Modified (Class E) condition during the current survey. The decline in the overall scores obtained at Sites MML1, MML2, MML3, MMD1 KAN and MMD2 KO is mainly a result of increased water quality modifications due to significant increases in EC levels.

The IHAS results indicate that the habitat integrity at Site MML2 is considered adequate in supporting a diverse macro-invertebrate community during the current assessment. The habitat integrity at the remainder of the sites is however inadequate in supporting a diverse community. The habitat integrity at Site MML2 has therefore improved from an inadequate state during the previous survey and has declined from an adequate state at Site MML3. The habitat integrity at the remainder of the sites has remained inadequate since April 2017. The habitat conditions on the Lilongwe River indicate some variation in habitat integrity between the sites, increasing in suitability from MML1 to MML3, after which a decreasing trend is evident further downstream at Sites MML4 and MML5. This is likely a result of the high water levels occurring within the system due to releases from the Kamuzu Reservoir, which is likely to have an influence on the structure of the aquatic communities present at these sites.

With regards to the SASS5 assessment, the macro-invertebrate integrity at Sites MML1, MML4, MML5, MMDR, MMD1 KAN and MMD2 KO is regarded as being in a Severely to Critically Modified (Class E/F) state and in a Largely Modified (Class D) state at Sites MML2 and MML3. Since the previous survey conducted in April 2017, the macro-invertebrate integrity has remained in a Severely to Critically Modified (Class E/F) state at Site MML1, has improved from a Severely to Critically Modified (Class E/F) state to Largely Modified (Class D) state at Site MML2, has declined from a Moderately Modified (Class C) state to a Largely Modified (Class D) state at Site MML3, has remained in a Severely to Critically Modified (Class E/F) state at Site MMDR, and has declined from a Largely Modified (Class D) state to a Severely to Critically Modified (Class E/F) state during the current survey at Sites MMD1 KAN and MMD2 KO.

Results from the diatom analysis indicate that the water quality at each site appears to have some pollution-related impacts and the overall water quality is Moderate for all sites. According to temporal diatom analysis trends, the ecological water quality has shown a general improvement since April 2017. The diatom assemblage at Site MML1 indicates alkaline, eutrophic conditions with an electrolyte-rich content and a low level of organic pollution. The level of organic pollution has decreased slightly since the previous survey in

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April 2017. The diatom community at Site MML2 points to meso- to eutrophic conditions with moderate electrolyte content and a relatively high level of organic pollution. The level of organic pollution increased slightly since the previous survey. The diatom assemblage at Site MML3 indicates eutrophic, moderate electrolyte freshwater conditions and a low level of organic pollution. The level of organic pollution at this site has decreased since the previous survey. The diatom community Site MML4 points to meso- to eutrophic conditions with moderate electrolyte content and appears to have a low impact associated with organic pollution. The diatom community at Site MML5 indicates slightly brackish, eutrophic conditions with electrolyte-rich content and no serious impact associated with organic pollution. The diatom community at Site MMDR reflects slightly brackish, meso- to eutrophic conditions with moderate electrolyte content and relatively low level of organic pollution. The overall ecological water quality shows a stable trend since the previous survey. The diatom community at Site MMD1 KAN points to alkaline, meso- to eutrophic conditions with moderate electrolyte content and no evidence of organic pollution. The overall water quality has improved since the previous survey. The diatom community at Site MMD2 KO indicates alkaline, eutrophic conditions with moderate electrolyte content and a relatively high level of organic pollution.

Results of the toxicological screening tests indicate that the water poses no acute hazard (Class I) to the aquatic communities at Sites MML1 to MML5 as well as at Site MMDR and the Kamuzu Reservoir II during the current assessment. The water at Sites MMD1 KAN and MMD2 KO, however, were found to pose a slight acute hazard (Class II). Since the previous survey in April 2017, the toxicological hazard has improved from a slight acute hazard (Class II) condition to a no acute hazard (Class I) condition at Site MML1, from an acute hazard (Class III) condition to a no acute hazard (Class I) condition at Sites MML2 and MML3 and from a high acute hazard (Class IV) condition to a no acute hazard (Class I) condition at Site MMDR during the current assessment. The toxicological hazard has remained in a slight acute hazard (Class II) condition at Sites MMD1 KAN and MMD2 KO. The levels of bacterial growth inhibition are low at all sites, with the exception of Sites MMD1 KAN and MMD2 KO, where bacterial growth inhibition is above 20%. The highest level of bacterial inhibition occurs at Site MMD1 KAN. Low levels of algal stimulation are evident at all sites. Low mortality rates of D. magna, which represents the macro-invertebrate community, are evident at Sites MML1, MMDR and MMD2 KO. Low mortality rates of P. retuculata, which represents the fish community, have also been recorded at Sites MML1 and MML4. This is supported by the metal analysis results that indicate a guideline exceedance in the lead (Pb) concentration measured in O. lidole from Site MML1 and the chromium (Cr) concentration measured in both A. calliptera and O. lidole from Site MML1 and in O. lidole from Site MML4.

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Results obtained from the wet (high flow) season survey indicate that cumulative impacts are likely to be occurring as a result of nutrient inputs within the Lilongwe River system and associated dambos, as well as the Lisungwi River system. This is likely to be attributed to the surrounding land-use including runoff from surrounding villages, agricultural fields containing fertilisers, livestock farming, the washing of garments and bathing within the streams. The release of phosphates, nitrates and ammonium into these systems is likely due to these activities resulting in a high potential for eutrophication (DWAF, 1996), which is likely to limit the colonisation of the aquatic biota over time through osmotic stress. An improvement in the level of organic pollution and toxicological hazard at Sites MML1 and MML3 on the Lilongwe River is however evident since the previous survey in April 2017 according to the current diatom analysis and toxicological testing results, respectively. Nevertheless, results from the baseline metal analysis indicate that some impact may be occurring within the fish community at Sites MML1 and MML4 due to elevated concentrations of lead (Pb) and chromium (Cr) measured in the fish tissue obtained from these sites, which is supported by the findings of the toxicological analysis, indicating the likelihood of low levels of fish mortality occurring at Sites MML1 and MML4. It is therefore essential that the concentration of metals within fish tissue be closely monitored at all applicable sites. Metal analysis within bivalve tissue (if possible, depending on availability) and sediment should also be conducted at all sites in order to provide a comprehensive representation of the metal concentrations occurring within the aquatic ecosystems in the vicinity of the Project area, particularly within the Kamuzu Reservoir II as elevated concentrations of chromium (Cr), copper (Cu), iron (Fe) and manganese (Mn) were measured within the sediment sampled at this point during the current assessment.

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6. RECOMMENDATIONS

The following is recommended as part of the current wet (high flow) season fish and mollusc baseline survey and aquatic biomonitoring assessment:

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8. APPENDICES

APPENDIX A: METAL ANALYSIS REPORT (FEBRUARY 2018)

17-1068 18 January 2019 Page 112 Date Date received: 2018/06/08 2018/08/14 Completed: Project number: 118 Report number: 75066

Client name: Groundwater Consulting Services Contact person: Sandra Carminati

Address: 63 Wessel Road, Rivonia, Johannesburg, South Africa Email: [email protected]

Telephone: +27 (0) 11 803 5726 Cell: + 27 (0) 83 451 3730

Fish Tissue

Sample Number (Client) Species Collection Site Date Sample Number Tab No

MML1 A1 Astatotilapia calliptera MML1 2018/02/18 32379 MML1 A2 Astatotilapia calliptera MML1 2018/02/18 32380 MML1 O1 Oreochromis lidole MML1 2018/02/18 32381 1 MML1 O2 Oreochromis lidole MML1 2018/02/18 32382 MML2 A1 Astatotilapia calliptera MML2 2018/02/18 32383 MML2 C1 Labeobarbus johnstonii MML2 2018/02/18 32384 MML3 A1 Astatotilapia calliptera MML3 2018/02/17 32385 MML3 A2 Astatotilapia calliptera MML3 2018/02/17 32386 MML4 A Astatotilapia calliptera MML4 2018/02/16 32387 2 MML4 O Oreochromis lidole MML4 2018/02/16 32388 MML4 Mollusc Coelatura mossambicensis MML4 2018/02/16 32389 D A1 Astatotilapia calliptera DAM 2018/02/17 32390 D A2 Astatotilapia calliptera DAM 2018/02/17 32391 3 D O11 Oreochromis lidole DAM 2018/02/17 32392 D O12 Oreochromis lidole DAM 2018/02/17 32393

Sediment

Sample Number (Client) Collection Site Date Sample Number Tab No

Dam Kamuzu Reservoir II 2018/02/17 32394 4

MML1 A1 Lab ID MML1 A2 Lab ID

Sample Name 32379 Sample Name 32380 Sample Weight 0.5005 Sample Weight 0.5001 Sample Volume 50 Sample Volume 50 Factor 100 Factor 100 Element Det Limit Element mg/l mg/kg Element mg/l mg/kg Al <0.100 Al 29 2878 Al 44 4387 As <0.010 As 0.006 0.620 As 0.006 0.628 Cd <0.010 Cd 0.000 0.000 Cd 0.000 0.000 Cr <0.010 Cr 0.045 4.45 Cr 0.026 2.60 Cu <0.010 Cu 0.042 4.17 Cu 0.046 4.64 Fe <0.025 Fe 27 2703 Fe 42 4184 Hg <0.010 Hg 0.000 0.000 Hg 0.000 0.000 Mn <0.025 Mn 1.15 115 Mn 1.35 135 Pb <0.010 Pb 0.005 0.476 Pb 0.007 0.747 Se <0.010 Se 0.016 1.56 Se 0.011 1.10

MML1 O1 Lab ID MML1 O2 Lab ID

Sample Name 32381 Sample Name 32382 Sample Weight 0.5000 Sample Weight 0.5003 Sample Sample Volume 50 Volume 50 Factor 100 Factor 100

Element Det Limit Element mg/l mg/kg Element mg/l mg/kg Al <0.100 Al 127 12676 Al 177 17686 As <0.010 As 0.017 1.70 As 0.017 1.75 Cd <0.010 Cd 0.000 0.000 Cd 0.000 0.000 Cr <0.010 Cr 0.065 6.49 Cr 0.112 11 Cu <0.010 Cu 0.169 17 Cu 0.236 24 Fe <0.025 Fe 126 12614 Fe 164 16434 Hg <0.010 Hg 0.000 0.000 Hg 0.000 0.000 Mn <0.025 Mn 3.53 353 Mn 4.35 435 Pb <0.010 Pb 0.024 2.39 Pb 0.031 3.08 Se <0.010 Se 0.008 0.770 Se 0.007 0.705

MML2 A1 Lab ID MML2 C1 Lab ID

Sample Name 32383 Sample Name 32384 Sample Weight 0.5005 Sample Weight 0.5001 Sample Volume 50 Sample Volume 50 Factor 100 Factor 100 Element Det Limit Element mg/l mg/kg Element mg/l mg/kg Al <0.100 Al 0.973 97.3 Al 0.702 70 As <0.010 As 0.001 0.113 As 0.001 0.078 Cd <0.010 Cd 0.000 0.000 Cd 0.000 0.000 Cr <0.010 Cr 0.005 0.546 Cr 0.002 0.154 Cu <0.010 Cu 0.023 2.32 Cu 0.032 3.15 Fe <0.025 Fe 2.13 212.5 Fe 1.14 114 Hg <0.010 Hg 0.000 0.000 Hg 0.000 0.000 Mn <0.025 Mn 0.627 62.7 Mn 0.227 23 Pb <0.010 Pb 0.001 0.102 Pb 0.000 0.000 Se <0.010 Se 0.014 1.39 Se 0.012 1.21

MML3 A1 Lab ID MML3 A2 Lab ID

Sample Name 32385 Sample Name 32386 Sample Weight 0.5000 Sample Weight 0.5000 Sample Volume 50 Sample Volume 50 Factor 100 Factor 100 Element Det Limit Element mg/l mg/kg Element mg/l mg/kg Al <0.100 Al 6.60 660 Al 3.12 312 As <0.010 As 0.003 0.323 As 0.003 0.275 Cd <0.010 Cd 0.000 0.000 Cd 0.000 0.000 Cr <0.010 Cr 0.018 1.75 Cr 0.009 0.931 Cu <0.010 Cu 0.023 2.31 Cu 0.015 1.47 Fe <0.025 Fe 7.47 747 Fe 3.51 351 Hg <0.010 Hg 0.000 0.000 Hg 0.000 0.000 Mn <0.025 Mn 2.54 254 Mn 1.61 161 Pb <0.010 Pb 0.001 0.137 Pb 0.001 0.068 Se <0.010 Se 0.007 0.661 Se 0.007 0.674

MML4 A Lab ID MML4 O Lab ID

Sample Name 32387 Sample Name 32388 Sample Weight 0.5005 Sample Weight 0.1268 Sample Volume 50 Sample Volume 25 Factor 100 Factor 197 Element Det Limit Element mg/l mg/kg Element mg/l mg/kg Al <0.100 Al 6.39 638.0 Al 35.3 3525.4 As <0.010 As 0.006 0.599 As 0.005 0.520 Cd <0.010 Cd 0.000 0.000 Cd 0.000 0.000 Cr <0.010 Cr 0.007 0.676 Cr 0.241 24.1 Cu <0.010 Cu 0.050 5.01 Cu 0.035 3.46 Fe <0.025 Fe 7.92 791.0 Fe 30.9 3085.1 Hg <0.010 Hg 0.000 0.000 Hg 0.000 0.000 Mn <0.025 Mn 8.64 862.8 Mn 1.42 141.6 Pb <0.010 Pb 0.001 0.129 Pb 0.006 0.550 Se <0.010 Se 0.002 0.196 Se 0.017 1.74

MML4 Mollusc Lab ID

Sample Name 32389

Sample Weight 0.4841

Sample Volume 50

Factor 103

Element Det Limit Element mg/l mg/kg

Al <0.100 Al 2.40 240.1

As <0.010 As 0.002 0.167

Cd <0.010 Cd 0.000 0.000

Cr <0.010 Cr 0.003 0.310

Cu <0.010 Cu 0.029 2.85

Fe <0.025 Fe 2.61 261.1

Hg <0.010 Hg 0.000 0.000

Mn <0.025 Mn 0.275 27.5

Pb <0.010 Pb 0.001 0.103

Se <0.010 Se 0.007 0.744

D A1 Lab ID D A2 Lab ID Sample Name 32390 Sample Name 31391 Sample Weight 0.5005 Sample Weight 0.5003 Sample Volume 50 Sample Volume 50 Factor 100 Factor 100 Element Det Limit Element mg/l mg/kg Element mg/l mg/kg Al <0.100 Al 1.31 131 Al 0.126 13 As <0.010 As 0.001 0.100 As 0.001 0.100 Cd <0.010 Cd 0.000 0.000 Cd 0.000 0.000 Cr <0.010 Cr 0.002 0.200 Cr 0.000 0.000 Cu <0.010 Cu 0.015 1.50 Cu 0.011 1.10 Fe <0.025 Fe 2.56 256 Fe 0.660 66 Hg <0.010 Hg 0.000 0.000 Hg 0.000 0.000 Mn <0.025 Mn 0.458 46 Mn 0.263 26 Pb <0.010 Pb 0.000 0.000 Pb 0.000 0.000 Se <0.010 Se 0.007 0.699 Se 0.009 0.899

D 011 Lab ID D 012 Lab ID Sample Name 31392 Sample Name 31393 Sample Weight 0.3847 Sample Weight 0.2471 Sample Volume 50 Sample Volume 25 Factor 130 Factor 101 Element Det Limit Element mg/l mg/kg Element mg/l mg/kg Al <0.100 Al 0.317 41 Al 2.72 276 As <0.010 As 0.001 0.130 As 0.002 0.202 Cd <0.010 Cd 0.000 0.000 Cd 0.000 0.000 Cr <0.010 Cr 0.000 0.000 Cr 0.001 0.101 Cu <0.010 Cu 0.017 2.21 Cu 0.046 4.65 Fe <0.025 Fe 1.32 172 Fe 3.64 368 Hg <0.010 Hg 0.000 0.000 Hg 0.000 0.000 Mn <0.025 Mn 0.447 58 Mn 0.987 100 Pb <0.010 Pb 0.000 0.000 Pb 0.006 0.607 Se <0.010 Se 0.005 0.650 Se 0.009 0.911

Dam (Sediment) Lab ID

Sample Name 32394 Sample Weight 0.2503 Sample Volume 100 Factor 400 Element Det Limit Element mg/l mg/kg Al <0.100 Al 133.4 53294.8 As <0.010 As 0.004 1.60 Cd <0.010 Cd 0.000 0.000 Cr <0.010 Cr 0.822 328.4 Cu <0.010 Cu 0.199 79.5 Fe <0.025 Fe 183.3 73250.5 Hg <0.010 Hg 0.000 0.000 Mn <0.025 Mn 3.09 1234.9 Pb <0.010 Pb 0.015 5.99 Se <0.010 Se 0.000 0.000

Fish Tissue Sample Number Species Collection Site Date Analysis No. MML1 A1 Astatotilapia calliptera MML1 2018/02/18 1 MML1 A2 Astatotilapia calliptera MML1 2018/02/18 2 MML1 O1 Oreochromis lidole MML1 2018/02/18 3 MML1 O2 Oreochromis lidole MML1 2018/02/18 4 MML2 A1 Astatotilapia calliptera MML2 2018/02/18 5 MML2 C1 Labeobarbus johnstonii MML2 2018/02/18 6 MML3 A1 Astatotilapia calliptera MML3 2018/02/17 Al, As, Cd, Cr, Cu, Fe, Pb, Mn, Se and Hg (As per quote). 7 MML3 A2 Astatotilapia calliptera MML3 2018/02/17 8 MML4 A Astatotilapia calliptera MML4 2018/02/16 9 MML4 O Oreochromis lidole MML4 2018/02/16 10 MML4 Mollusc Coelatura mossambicensis MML4 2018/02/16 11 D A1 Astatotilapia calliptera DAM 2018/02/17 12 D A2 Astatotilapia calliptera DAM 2018/02/17 13 D O11 Oreochromis lidole DAM 2018/02/17 14 Al, As, Cd, Cr, Cu, Fe, Pb, Mn, Se and Hg (As per quote). D O12 Oreochromis lidole DAM 2018/02/17 15 Sediment Sample Number Collection Site Date Analysis No. Dam Dam 2018/02/17 Al, As, Cd, Cr, Cu, Fe, Pb, Mn, Se and Hg (as per quote). 16

Sovereign Metals Limited Aquatic Biomonitoring Assessment

APPENDIX B: DIATOM ANALYSIS REPORT (FEBRUARY 2018)

17-1068 18 January 2019 Page 123 Diatom Analysis Report June 2018

GCS- Malawi Project Diatom Analysis Report

Reference: GCS_Malawi_Diatoms _June_2018 Date: June 2018 No. of samples: Eight Version: Final

GCS- Malawi Project - 1 - Diatom Analysis Report June 2018

Prepared For: Prepared By:

GCS Water and Environmental Consultants Ecotone Freshwater Consultants 63 Wessel Road P.O Box 84, Florida, 1710 Rivonia, 2128 (T): 011 672 1375 Johannesburg, South Africa (C): +27 84 585 7479 (T): +27 (0) 11 803 5726 (F): 088 011 673 1192 (F): +27 (0) 803 5745 [email protected] Sandra Carminati www.ecotone-sa.co.za [email protected] [email protected]

GCS- Malawi Project - 2 - Diatom Analysis Report June 2018

Report Author

Professional Registration - Person Qualifications Report Status SACNASP

Report compiled Diatomologist Megan Gomes MSc (Ecology) Wits’15 First Draft by Pr. Sci. Nat. (Pending)

Members: Michiel Jonker & Marco Alexandre Registration no: CK 2008/027022/23

Report Checked By

______

Full Name: Marco Alexandre Title / Position: Aquatic Ecologist and Partner Qualification(s): M.Sc. (Aquatic Health) Registration: Pri. Sci. Nat. (400079/13)

______

Full Name: Michiel Jonker Title / Position: Aquatic Ecologist and Partner Qualification(s): M.Sc. (Aquatic Health), M.Sc. (Environmental Management) Registration: Pri. Sci. Nat. (400275/12)

GCS- Malawi Project - 3 - Diatom Analysis Report June 2018

Limitations and Disclaimer

The spatial and temporal extents of Ecotone Freshwater Consultants CC (Ecotone) services are described in the proposal and are subject to restrictions and limitations. A total assessment of all probable scenarios or circumstances that may exist on the study site was not undertaken. No assumptions should be made unless opinions are specifically indicated and provided. Data presented in this document may not elucidate all possible conditions that may exist given the limited nature of the enquiry.

Ecotone exercises reasonable skill, care and diligence in the provision of services, however, Ecotone accepts no liability or consequential liability for the use of the supplied project deliverables (in part or whole) and any information or material contained therein. The client, including their agents, by receiving these deliverables, indemnifies Ecotone (including its members, employees, and sub- consultants) against any actions, claims, demands, losses, liabilities, costs, damages and expenses arising directly or indirectly from or in connection with services rendered, directly or indirectly by Ecotone.

The project deliverables, including the reported results, comments, recommendations, and conclusions, are based on the author/s professional knowledge as well as available information. Ecotone, therefore, reserves the right to modify aspects of the project deliverables if and when new/additional information becomes available from research or further work in the applicable field of practice or about this study. Ecotone also reserves the right to authorise peer review of this deliverable by an independent third party.

GCS- Malawi Project - 4 - Diatom Analysis Report June 2018

Declaration of Independence

I, Marco Alexandre, as duly authorised representative of Ecotone Freshwater Consultants CC (Ecotone), hereby confirm my independence (as well as that of Ecotone) as a specialist and declare that neither I nor Ecotone have any interest, be it business, financial, personal or other, in any proposed activity, application or appeal, other than fair remuneration for work performed, specifically in connection with the diatom assessment for GCS- Malawi sites.

______

Full Name: Marco Alexandre Title / Position: Aquatic Ecologist and Partner Qualification(s): M.Sc. (Aquatic Health) Registration: Pri. Sci. Nat. (400079/13))

GCS- Malawi Project - 5 - Diatom Analysis Report June 2018

Table of Contents

Table of Contents ...... - 6 - List of Tables ...... - 6 - List of Figures ...... - 6 - List of Abbreviations ...... - 7 - Key Terminology outlined in (Taylor et al., 2007a) ...... - 7 - Executive summary ...... - 8 - 1. Introduction and Scope of Work ...... - 9 - 2. Methodology ...... - 9 - 3. Results and Discussion ...... - 10 - 3.1. Spatial Diatom Analysis ...... - 10 - 3.2. Temporal Diatom Trend Analysis ...... - 17 - 4. Summary and Conclusions ...... - 18 - 5. References ...... - 19 -

List of Tables

Table 2-1: Class values used for the Specific Pollution Index and Biological Diatom Index in the evaluation of water quality (adapted from Eloranta & Soininen, 2002) ...... - 10 -

Table 2-2: Interpretation of the percentage Pollution Tolerant Valves scores (adapted from Kelly, 1998)...... - 10 -

Table 3-2: Species and their abundances for the eight sites for February 2018 ...... - 15 -

Table 3-3: Diatom index scores for the study sites indicating the ecological water quality for February 2018 ...... - 17 -

Table 3-4: Diatom index scores for the study sites indicating the ecological water quality trends between seasons ...... - 17 -

List of Figures

Figure 3.1: Dominant diatom species recorded at all sites included: A, B) Nitzschia sp. and C) Gomphonema parvulum (Kelly et al., 2005)...... - 11 -

GCS- Malawi Project - 6 - Diatom Analysis Report June 2018

List of Abbreviations

BDI Biological Diatom Index %PTV Percentage Pollution Tolerant Valves SPI Specific Pollution Sensitivity Index

Key Terminology outlined in (Taylor et al., 2007a)

Trophy Description

Rich in organic matter, usually in the form of suspended plant colloids, but of a low Dystrophic nutrient content. Low levels or primary productivity, containing low levels of mineral nutrients required by Oligotrophic plants. Intermediate levels of primary productivity, with intermediate levels of mineral nutrients Mesotrophic required by plants.

Eutrophic High primary productivity, rich in mineral nutrients required by plants.

Very high primary productivity, constantly elevated supply of mineral nutrients required Hypereutrophic by plants.

Mineral Content Value

Very electrolyte poor < 50 μS/cm Electrolyte-poor (low electrolyte content) 50 - 100 μS/cm Moderate electrolyte content 100 - 500 μS/cm Electrolyte-rich (high electrolyte content) > 500 μS/cm Brackish (very high electrolyte content) > 1000 μS/cm Saline 6000 μS/cm

Pollution (Saprobity) Value

Unpolluted to slightly polluted (oligosaprobic) BOD <2, O2 deficit <15%

Moderately polluted (-mesosaprobic) BOD <4, O2 deficit <30%

Critical level of pollution (-á-mesosaprobic) BOD <7(10), O2 deficit <50%

Strongly polluted (á-mesosaprobic) BOD <13, O2 deficit <75%

Very heavily polluted (polysaprobic) BOD <22, O2 deficit <90%

GCS- Malawi Project - 7 - Diatom Analysis Report June 2018

Executive summary

Diatom laboratory procedures were carried out according to the methodology described by Taylor et al. (2005). The Percentage of Pollution Tolerant Valves (%PTV; Kelly & Whitton, 1995) and ecological descriptor indices (Van Dam et al., 1994) were included in the analysis to indicate organic pollution. A total of 65 diatom species were recorded at the eight Malawi sites (MML1, MML2, MML3, MML4, MML5, MMDR, MMD1 KAN and MMD2 KO). The diatom assemblages were generally comprised of species characteristic of fresh-brackish, circumneutral to alkaline waters and eutrophic conditions. The pollution levels indicated that there was some form of pollution evident at all the sites. Based on the diatom community analyses, the water quality at each site appeared to have some pollution related impacts and the overall water quality was Moderate for all sites. The organic or anthropogenic pollution related impacts may be attributed to the surrounding land-use. According to temporal diatom analysis trends the ecological water quality has shown a general improvement from the April 2017 to the February 2018 survey and a stable trend between the previous October 2017 and the current February 2018 survey.

GCS- Malawi Project - 8 - Diatom Analysis Report June 2018

1. Introduction and Scope of Work

2. Methodology

Diatom laboratory procedures were carried out according to the methodology described by Taylor et al. (2005). Diatom samples were prepared for microscopy by using the hot hydrochloric acid and potassium permanganate method (Taylor et al., 2007a). Approximately 300 to 400 diatom valves were identified and counted to produce semi-quantitative data for analysis. Prygiel et al. (2002) found that diatom counts of 300 valves and above were necessary to make correct environmental inferences. The taxonomic guide by Taylor et al. (2007b) and Cantonati et al. (2017) was consulted for identification purposes. Where necessary, Krammer & Lange-Bertalot (1986, 1988, 1991 a, b) were used for identification and confirmation of species identification. Environmental preferences were inferred from Taylor et al. (2007b) and Cantonati et al. (2017) and various other literature sources as indicated in the discussion section to describe the environmental water quality at each site.

Two indices, namely the Specific Pollution Sensitivity Index (SPI; CEMAGREF, 1982) and the Biological Diatom Index (BDI; Lenoir & Coste, 1996) were used in the diatom assessment (Table 2-1). The SPI is an inclusive index and takes factors such as salinity, eutrophication and organic pollution into account. This index comprises 2035 taxa (Taylor, 2004) and is recognised as the broadest species base of any index currently in use and has been adapted to include taxa endemic to and commonly found in South Africa, thus increasing the accuracy of diatom-based water quality assessments and is known as the South African Diatom Index (SADI) (Harding and Taylor, 2011). The limit values and associated ecological water quality classes adapted from Eloranta & Soininen (2002) were used for interpretation of the SPI and BDI scores. The SPI and BDI are based on a score between 0 – 20, where a score of 20 indicates no pollution and a score of zero indicates an increasing level of pollution or eutrophication.

GCS- Malawi Diatoms - 9 - Diatom Analysis Report June 2018

Table 2-1: Class values used for the Specific Pollution Index and Biological Diatom Index in the evaluation of water quality (adapted from Eloranta & Soininen, 2002)

Index Score Class

>17 High quality 13 to 17 Good quality 9 to 13 Moderate quality 5 to 9 Poor quality <5 Bad quality

In addition, the Percentage of Pollution Tolerant Valves (%PTV; Kelly & Whitton, 1995) and ecological descriptor indices (Van Dam et al., 1994) were used to indicate organic pollution (Table 2-2). The Percentage Pollution Tolerant Valves (%PTV) is part of the UK Trophic Diatom Index (TDI) (Kelly and Whitton, 1995) and was developed for monitoring possible impacts of organic pollution (sewage outfall- orthophosphate-phosphorus concentrations), and not general stream quality. The %PTV has a maximum score of 100, where a score of zero indicates no organic pollution and a score of 100 indicates definite and severe organic pollution. The presence of more than 21% PTVs shows organic impact. All calculations were computed using OMNIDIA ver. 4.2 program (Lecointe et al., 1993).

Table 2-2: Interpretation of the percentage Pollution Tolerant Valves scores (adapted from Kelly, 1998)

%PTV Interpretation

<20 Site free from organic pollution. 21 to <40 There is some evidence of organic pollution. 41 to 60 Organic pollution likely to contribute significantly to eutrophication. >61 Site is heavily contaminated with organic pollution.

3. Results and Discussion

3.1. Spatial Diatom Analysis

A total of 65 diatom species were recorded at the eight sites and the dominant diatom species recorded at all sites included, Nitzschia sp. and Gomphonema parvulum ( Figure 3.1). It is important to know that these dominant species are cosmopolitan and have very wide ecological amplitudes, thus caution must be taken when analysing the predominance of these

GCS- Malawi Diatoms - 10 - Diatom Analysis Report June 2018

species at specific sites and it is important to consider the diatom assemblage as a whole in conjunction with focusing on the dominant species. Nitzschia sp. pointed to α-mesosaprobic to polysaprobic freshwater and is commonly found in untreated waste water and in habitats that are strongly impacted by industrial sewerage. Gomphonema parvulum indicated oligosaprobic and mesosaprobic, oligo- to eutrophic freshwater.

Figure 3.1: Dominant diatom species recorded at all sites included: A, B) Nitzschia sp. and C) Gomphonema parvulum (Kelly et al., 2005).

Additional information is provided for the sub-dominant species in order to make ecological inferences for the eight sites assessed (Table 3-1, Table 3-2; Taylor et al., 2007, Cantonati et al., 2017):

Site MML1: This site was dominated by Nitzschia sp. which pointed to eutrophic, polluted (α-mesosaprobic to polysaprobic) freshwater. The dominance of Navicula sp. suggested alkaline, low temperature, eutrophic running water with medium-high conductivity and species from this genus are commonly found in organically polluted water. The presence of G. parvulum indicated impacts associated with agricultural run-off and is adapted to withstand physical disturbance and benefits from organic enrichment. The presence of Orthoseira roeseana pointed to aerial habitats indicating that this site may experience periodic drying out. The presence of Gomphonema sp. can be found in clean waters as well as water receiving sewage effluent. The diatom assemblage at this site indicated alkaline, eutrophic conditions with electrolyte-rich content. Owing to the presence of the dominant species, this site appeared to be impacted by organic/anthropogenic pollution which is

GCS- Malawi Diatoms - 11 - Diatom Analysis Report June 2018

possibly associated with the surrounding land-use. The %PTV score was relatively low indicating that there was no serious impact associated with organic pollution at this site. The level of organic pollution showed a slight decrease since the previous survey and the overall water quality appeared to remain stable and considered Moderate (Table 3-2). Site MML2: The overwhelming dominance of G. parvulum and G. rhombicum indicated oligosaprobic and mesosaprobic, oligo- to eutrophic, electrolyte-poor freshwater and pointed to impacts associated with agricultural run-off. The subdominance of Nitzschia sp. pointed to α-mesosaprobic to polysaprobic freshwater and this taxon is commonly found in untreated waste water and in habitats that are strongly impacted by industrial sewerage. The subdominant, N. symmetrica and Navicula sp. pointed to eutrophic, electrolyte-rich waters and both taxa are tolerant of strongly polluted conditions. The presence of Achananthidum sp. has been recorded in high proportions over a wide range of trophic levels and is usually absent from moderately- to strongly-acidic or very electrolyte-poor environments. The diatom community at this site pointed to meso- to eutrophic conditions with moderate electrolyte content and owing to the presence of the dominant species there appeared to be some form of organic pollution impacts at this site. The %PTV score was relatively high indicating that there was some impact associated with organic pollution, possibly originating from the surrounding land-use. The level of organic pollution showed a slight increase since the previous survey, however despite this the overall water quality was considered Moderate (Table 3-2). Site MML3: The dominance of Achnanthidium sp. indicated clean and polluted waters, including those affected by acid mine drainage (Ponader & Potapova, 2007) and is usually absent from moderately- to strongly-acid or very electrolyte poor environments. The subdominance of Nitzschia sp. and N. amphibia pointed to eutrophic freshwater and are tolerant to strongly polluted conditions. In particular, Nitzschia sp. are commonly found in untreated waste water and in habitats that are strongly impacted by industrial sewerage. The presence of Amphora veneta indicated elevated electrolyte content, tolerating heavy pollution. The presence of G. parvulum indicated oligosaprobic and mesosaprobic, oligo- to eutrophic freshwater and pointed to impacts associated with agricultural run-off. The presence of Cyclotella meneghiniana pointed to eutrophic, electrolyte rich rivers. The diatom assemblage at this site indicated eutrophic, moderate electrolyte freshwater conditions. The %PTV score was relatively low but owing to the presence of Nitzschia sp. this site appeared to be slightly impacted by some form of organic/anthropogenic pollution

GCS- Malawi Diatoms - 12 - Diatom Analysis Report June 2018

entering the system. The level of organic pollution decreased since the previous survey, but the overall water quality remained stable and was considered Moderate (Table 3-2). Site MML4: This site was dominated by Achnanthidium sp. which pointed to calcium- bicarbonate rich, mesotrophic to eutrophic freshwater with medium-high conductivity. The subdominance of Nitzschia sp. and N. amphibia pointed to eutrophic freshwater and are tolerant to strongly polluted conditions. The subdominance of Cyclotella meneghiniana pointed to eutrophic, electrolyte rich rivers. The presence of Navicula sp. suggested alkaline, low temperature, eutrophic running water with medium-high conductivity and species from this genus are commonly found in organically polluted water. The presence of Ulnaria ulna indicated alkaline, medium conductivity, oligosaprobic, moderately eutrophic habitat. The diatom community at this site pointed to meso- to eutrophic conditions with moderate electrolyte content. Owing to the presence of Achnanthidium sp. and the relatively low %PTV score at this site appeared to have a low impact associated with organic pollution. The overall water quality was considered Moderate (Table 3-2). Site MML5: This site was dominated by Navicula sp. which suggested alkaline, low temperature, eutrophic running water with medium-high conductivity and species from this genus are commonly found in organically polluted water. The subdominance of Cyclotella meneghiniana pointed to eutrophic, electrolyte rich rivers. The subdominance of Orthoseira roeseana and Navicula erifuga pointed to brackish conditions and both species are adapted to intermittently wet conditions. The presence of Nitzschia sp. pointed to eutrophic, polluted (α-mesosaprobic to polysaprobic) freshwater. The diatom community at this site pointed to slightly brackish, eutrophic conditions with electrolyte-rich content. The %PTV score was low indicating that there was no serious impact associated with organic pollution and the overall water quality was considered Moderate (Table 3-2). Site MMDR: This site was dominated by Nitzschia sp. and N. sigma which pointed to eutrophic, electrolyte-rich and brackish conditions. The subdominance of Pinnularia gibba pointed to calcium-bicarbonate-poor freshwater and may occur in rivers strongly impacted by waste water. The presence of Gyrosigma acuminatum indicated alkaline freshwater with high trophic levels and sensitive to strongly polluted conditions. The presence of Frustulia crassinervia pointed to dystrophic, electrolyte-poor freshwater and this taxon is usually an indicator of good ecological quality. The presence P. subcapitata pointed to oligotrophic, electrolyte-poor and weakly acidic conditions. The occurrence of Navicula sp. suggested alkaline, low temperature, eutrophic running water with medium-high conductivity and species from this genus are commonly found in organically polluted water. The diatom

GCS- Malawi Diatoms - 13 - Diatom Analysis Report June 2018

community at this site pointed to slightly brackish, meso- to eutrophic conditions with moderate electrolyte content. The %PTV score was relatively low indicating that there was no serious impact associated with organic pollution. The overall ecological water quality showed a stable trend since the previous survey and was considered Moderate (Table 3-2). Site MMD1 KAN: The overwhelming dominance of Nitzschia sp. pointed to eutrophic, polluted (α-mesosaprobic to polysaprobic) freshwater. The subdominance of Pinnularia marchica pointed to alkaline, strongly eutrophic, moderately polluted running waters with medium electrolyte content, particularly developed under conditions of cold water temperature. The presence of Pinnularia sp. pointed to calcium-bicarbonate-poor freshwater and may occur in rivers strongly impacted by waste water. The presence of Stauroneis anceps pointed to oligo- to eutrophic, but oligosaprobic freshwater with medium electrolyte content. The presence of P. borealis indicated pseudaerial habitats, such as soils, bryophyte carpets and rock walls. The diatom community at this site pointed to alkaline, meso- to eutrophic conditions with moderate electrolyte content and owing to the presence of Nitzschia there appeared to be some form of pollution impact at this site. The %PTV score indicated that there was no evidence of organic pollution suggesting that some other form of pollution was impacting this site. The overall water quality was considered Moderate and showed an improvement since the previous survey (Table 3-2). Site MMD2 KO: The overwhelming dominance of Nitzschia sp. pointed to eutrophic, polluted (α-mesosaprobic to polysaprobic) freshwater. The subdominance of G. parvulum and Gomphonema sp. indicated oligosaprobic and mesosaprobic, oligo- to eutrophic, electrolyte-poor freshwater and pointed to impacts associated with agricultural run-off. The subdominance of Adlafia bryophila pointed to a broad electrolyte content range and this taxon is sensitive to organic pollution. The presence of Pinnularia sp. pointed to calcium- bicarbonate-poor freshwater and may occur in rivers impacted by waste water. The presence of Sellaphora pupula indicated alkaline, eutrophic to polytrophic conditions with medium to increased electrolyte content. The diatom community at this site pointed to alkaline, eutrophic conditions with moderate electrolyte content. Owing to the dominance of Nitzschia sp. and the relatively high %PTV score this site appeared to be impacted by some form of organic/anthropogenic pollution. The overall water quality was considered Moderate (Table 3-2).

GCS- Malawi Diatoms - 14 - Diatom Analysis Report June 2018

Table 3-1: Species and their abundances for the eight sites for February 2018

MMD1 MMD2 Taxa MML1 MML2 MML3 MML4 MML5 MMDR KAN KO

Achnanthidium sp. 40 78 64 7 5 10 Adlafia bryophila (Petersen) Moser Lange- 7 5 4 36 Bertalot & Metzeltin Amphora veneta 14 13 30 10 3 4

Aulacoseira granulata 16 20 28 2

Caloneis fontinalis (Grun.) Lange-Bertalot & 4 Reichardt Caloneis lancettula (Schulz) Lange-Bertalot 4 4 & Witkowski Cocconeis placentula Ehrenberg var. 6 8 2 placentula Craticula ambigua (Ehrenberg) Mann 6

Craticula buderi (Hustedt) Lange-Bertalot 0 8 11

Craticula molestiformis (Hustedt) Lange- 20 7 15 Bertalot Craticula minusculoides (Hustedt) Lange- 10 18 6 16 Bertalot Cyclotella meneghiniana Kützing 18 16 30 56 4 4

Cymbella tumida (Brebisson)Van Heurck 6 6 3

Cymbella turgidula Grunow 1875 in 2 3 A.Schmidt & al. var. turgidula Diadesmis confervacea Kützing var. 20 confervacea Encyonema minutum 12 10 4 10 10 Encyonema silesiacum (Bleisch in Rabh.) 14 4 12 D.G. Mann Epithemia sorex Kützing 4

Eunotia botuliformis Wild Norpel & Lange- 4 4 Bertalot Eunotia minor (Kützing) Grunow in Van 4 Heurck Eunotia sp. 4

Fragilariforma virescens 14

Fragilaria dilatata (Brébisson) Lange- 22 Bertalot Fragilaria species 8 42 15 38 4 4

Frustulia asiatica (skvortzow) Metzeltin 4 5 Lange-Bertalot & Nergui Frustulia crassinervia (Breb.) Lange- 7 7 3 36 Bertalot et Krammer Gomphonema minutum(Ag.)Agardh f. 20 4 minutum Gomphonema parvulum 26 110 15 16 6 78 Gomphonema rhombicum M. Schmidt 35 18

Gomphonema species 30 12 25 20 12 6 4 25 Gyrosigma acuminatum 10 5 5 3 8 34 5 (Kützing)Rabenhorst Hippodonta capitata 4 6

Lemnicola hungarica (Grunow) Round & 4 Basson Luticola species 4

GCS- Malawi Diatoms - 15 - Diatom Analysis Report June 2018

MMD1 MMD2 Taxa MML1 MML2 MML3 MML4 MML5 MMDR KAN KO

Navicula erifuga Lange-Bertalot 12 17 28

Navicula radiosa 12 17 28 3

Navicula rostellata 5 10

Navicula sp. 86 22 7 28 68 5 4 12 Navicula symmetrica Patrick 39 12 19

Neidium affine(Ehrenberg)Pfitzer 4 15 4

Neidium bisulcatum (Lagerstedt) Cleve 4

Nitzschia amphibia 10 40 23 10 10

Nitzschia sigma (Kützing)W.M.Smith 9 28 7 8 38 Nitzschia sp.1 88 35 48 50 50 48 168 108 Orthoseira roeseana (Rabenhorst) O'Meara 32 4 20 36 5

Pinnularia obscura Krasske 4

Pinnularia rhombarea Krammer 12 0 var.halophila Krammer Pinnularia borealis Ehrenberg var. borealis 4 30 10

Pinnularia gibba Ehrenberg 7 42 42 4

Pinnularia marchica Ilka Schönfelder var. 0 94 marchica Pinnularia obscura Krasske 25

Pinnularia sardoelliptica Lange-Bertalot & 13 32 Metzeltin Pinnularia subcapitata Gregory var. 24 subcapitata Placoneis symmetrica (Hustedt) Lange- 12 12 8 Bertalot Placoneis undulata 4

Planothidium frequentissimum(Lange- 4 Bertalot)Lange-Bertalot Sellaphora pseudopupula (Krasske) Lange- 4 Bertalot Sellaphora pupula (Kützing) 4 4 2 22 Mereschkowksy Sellaphora species 11

Stauroneis anceps Ehrenberg 6 28

Surirella minuta Brebisson 4 4

Surirella sp.1 6

Surirella terricola Lange-Bertalot & Alles 14 4 6

Tabularia fasciculata (Agardh)Williams et 6 Round Ulnaria ulna (Nitzsch.) Compère 18 7 10 3 4

Total 450 450 450 450 450 280 450 450 Nutrients Salinity Organics pH-Alkaline pH-Slightly Acidic Other Dominant

GCS- Malawi Diatoms - 16 - Diatom Analysis Report June 2018

Table 3-2: Diatom index scores for the study sites indicating the ecological water quality for February 2018

Site %PTV SPI BDI Quality

3.2. Temporal Diatom Trend Analysis

It is important to monitor temporal trends in the diatom community in order to determine any changes in the ecological conditions of the aquatic environment and the associated impacts if any. According to temporal diatom analysis trends the ecological water quality has shown a general improvement from the April 2017 to the February 2018 surveys (Table 3-3). In comparison to the previous October 2017 survey, Site MMD1 KAN showed an improvement in water quality from Poor to Moderate. This slight improvement is likely associated with the decrease in organic pollution at this site. The water quality at the remainder of the sites showed a stable trend, despite the slight decrease in organic pollution. Site MML2 showed a slight increase in organic pollution but the level was low indicating that there were no serious impacts at this site.

Table 3-3: Diatom index scores for the study sites indicating the ecological water quality trends between seasons

Quality Quality Quality Site Trend April 2017 October 2017 February 2018 MML1 Bad Moderate Moderate ► MML2 Bad Moderate Moderate ► MML3 Bad Moderate Moderate ► MMD1 KAN Bad Poor Moderate ▲ MMDR Bad Moderate Moderate ►

GCS- Malawi Diatoms - 17 - Diatom Analysis Report June 2018

4. Summary and Conclusions

The diatom findings are summarised below:

The diatom assemblages were generally comprised of species characteristic of fresh-brackish, circumneutral to alkaline waters and eutrophic conditions. The pollution levels indicated that there was some form of pollution evident at all the sites. Based on the diatom community analyses, the water quality at each site appeared to have some pollution related impacts and the overall water quality for all sites was Moderate. The organic or anthropogenic pollution related impacts may be attributed to the surrounding land-use. According to temporal diatom analysis trends the ecological water quality has shown a general improvement from the April 2017 to the February 2018 surveys and a stable trend between the previous October 2017 and the current February 2018 survey.

GCS- Malawi Diatoms - 18 - Diatom Analysis Report June 2018

5. References

Besse-Lototskaya, A., Verdonschot, P. F. M., Coste, M., Van de Vijver, B. (2011). Evaluation of European diatom trophic indices. Ecological Indicators, 11, 456-467. Cantonati, M., Kelly, M.G. and Lange-Bertalot, H. (2017). Freshwater benthic diatoms of central Europe: Over 800 common species used in ecological assessment. Koeltz Botanical Books. CEMAGREF. (1982). Etude des méthodes biologiques quantitatives d'appréciation de la qualité des eaux. Rapport Division Qualité des Eaux Lyon - Agence Financiére de Bassin Rhône- Méditerranée- Corse. Pierre-Benite. Eloranta, P. & Soininen, J. (2002). Ecological status of Finnish rivers evaluated using benthic diatom communities. Journal of Applied Phycology, 14: 1-7. Kelly, M.G. & Whitton, B.A. (1995). The trophic diatom index: a new index for monitoring eutrophication in rivers. Journal of Applied Phycology, 7: 433-444. Kelly, M.G. (1998) Use of the Trophic Diatom Index to monitor eutrophication in rivers. Water Research, 32: 236-242. Kelly, M.G., Bennion, H., Cox, E.J., Goldsmith, B.m, Jamieson, J., Juggins, S., Mann, D.G & Telford, R.J. (2005). Common freshwater diatoms of Britain and Ireland: an interactive key. Environment Agency, Bristol. Retrieved from (http://craticula.ncl.ac.uk/EADiatomKey/html/ taxon13410310.html). Krammer, K. & Lange-Bertalot, H. (1986). Bacillariophyceae.1. Teil: Naviculaceae. In: Suβwasserflora von Mittleuropa, Band 2/1. Edited by Ettl, H., Gerloff, J., Heynig, H. & Mollenhauer, D. Spektrum Akademischer Verlag, Heidelberg, Berlin. Krammer, K. & Lange-Bertalot, H. (1988). Bacillariophyceae. 2. Teil: Bacillariaceae, Epithemiaceae, Surirellaceae. In: Suβwasserflora von Mittleuropa, Band 2/2. Edited by Ettl, H., Gerloff, J., Heynig, H. & Mollenhauer, D. Spektrum Akademischer Verlag, Heidelberg, Berlin. Krammer, K. & Lange-Bertalot, H. (1991a). Bacillariophyceae. 3. Teil: Centrales, Fragilariaceae, Eunotiaceae. In: Suβwasserflora von Mittleuropa, Band 2/3. Edited by Ettl, H., Gerloff, J., Heynig, H. & Mollenhauer, D. Spektrum Akademischer Verlag, Heidelberg, Berlin. Krammer, K. & Lange-Bertalot, H. (1991b). Bacillariophyceae. 4. Teil: Achnanthaceae, Kritische Erg¨anzungen zu Navicula (Lineolatae und Gomphonema). In: Suβwasserflora von Mittleuropa, Band 2/2. Edited by Ettl, H., Gerloff, J., Heynig, H. & Mollenhauer, D. Spektrum Akademischer Verlag, Heidelberg, Berlin. Lecointe, C., Coste, M. & Prygiel, J. (1993). “Omnidia”: Software for taxonomy, calculation of diatom indices and inventories management. Hydrobiologia 269/270: 509-513.

GCS- Malawi Diatoms - 19 - Diatom Analysis Report June 2018

Lenoir, A. & Coste, M. (1996). Development of a practical diatom index of overall water quality applicable to the French National Water Board network. In Use of Algae for Monitoring Rivers II: Edited by Whitton, B.A. & Rott, E. Institut für Botanik, Universität Innsbruck. pp. 29-43. Ponader, K. C., & Potapova, M. G. (2007). Diatoms from the genus Achnanthidium in flowing waters of the Appalachian Mountains (North America): ecology, distribution and taxonomic notes. Limnologica, 37: 227–241. Prygiel, J., Carpentier, P., Almeida, S., Coste, M., Druart, J.C., Ector, L., Guillard, D., Honeré, M.A., Iserentant, R., Ledeganck, P., Lalanne-Cassou, C., Lesniak, C., Mercier, I., Moncaut, P., Nazart, M., Nouchet, N., Peres, F., Peeters, V., Rimet, F., Rumeau, A., Sabater, S., Straub, F., Torrisi, M., Tudesque, L., van der Vijver, B., Vidal, H., Vizinet, J. & Zydek, N. (2002). Determination of the biological diatom index (IBD NF T 90-354): Results of an intercomparison exercise. Journal of Applied Phycology, 14: 27-39. Szczepocka E. (2007). Benthic diatoms from the outlet section of the Bzura River 30 years ago and presently. Oceanological and Hydrobiological Studies, 36: 255-260. Taylor, J.C., De la Rey, A. and Van Rensburg, L. (2005) Recommendations for the collection, preparation and enumeration of diatoms from riverine habitats for water quality monitoring in South Africa. African Journal of Aquatic Science, 30(1): 65–75. Taylor, JC, Harding, WR and Archibald, CGM (2007a). A methods manual for the collection, preparation and analysis of diatom samples. Water Research Commission Report TT281/07. Water Research Commission. Pretoria. Taylor, J.C., Harding, W.R. & Archibald, C.G.M. (2007b). An illustrated guide to some common diatom species from South Africa. WRC Report No. TT 282/07. Water Research Commission, Pretoria, South Africa. Van Dam, H., Mertens, A. & Sinkeldam, J. (1994). A coded checklist and ecological indicator values of freshwater diatoms from the Netherlands. Netherlands Journal of Aquatic Ecology, 28: 133-17. Zelinka, M. & Marvan, P. (1961). Zur Präzisierung der biologischen klassifikation der Reinheit flieβender Gewässer. -Arch. Hydrobiol., 57: 389-407.

GCS- Malawi Diatoms - 20 - Project Name Sample ID Substrate Type Date Sample Collected Collector Date Sample Preserved Preserved with Malawi MML1 Lilies 2018/02/14 Sandra 2018/02/14 70% ETOH Malawi MML2 Stones 2018/02/15 Sandra 2018/02/15 70% ETOH Malawi MML3 Stones 2018/02/14 Sandra 2018/02/14 70% ETOH Malawi MML4 Reed Stems 2018/02/16 Sandra 2018/02/16 70% ETOH Malawi MML5 Reed Stems 2018/02/16 Sandra 2018/02/16 70% ETOH Malawi MMDR Reeds Stems 2018/02/16 Sandra 2018/02/16 70% ETOH Malawi MMD1 Kan Seeds 2018/02/17 Sandra 2018/02/17 70% ETOH Malawi MMD2 KO Reed Stems 2018/02/17 Sandra 2018/02/17 70% ETOH Sovereign Metals Limited Aquatic Biomonitoring Assessment

APPENDIX C: TOXICOLOGICAL REPORT (FEBRUARY 2018)

17-1068 18 January 2019 Page 145

GAUTENG OFFICE: LOWVELD OFFICE: P.O. Box 11216, Silver Lakes, PO Box 1358, Malelane, 1320 Pretoria, 0054 Plot 356A, Zwavelpoort, Pretoria www.biotoxsa.co.za Fax: 086 535 7368 Fax: 086 628 6926 Lizet Moore: 082 554 4857 / Pieter Kotze: 082 890 6452 T0663 Company registration number: 2012/106020/07 012 753 2192 VAT number: 4740264959 Email: [email protected] [email protected]

Toxicity Specialists

TOXICITY TEST REPORT

For: GCS Water & Environment

63 Wessel Road, Rivonia, 2128 PO Box 2597, Rivonia, 2128

Survey: 2018-02

Report reference: GCS-C-18_TOX

Revision: 0

Project: Malingunde

Samples: MMD1 KAN, MMD2 KO, MML1, MML2, MML3, MML4, MML5, MMDR, DAM

Tests performed by: Marrilize Bylsma (Technical Manager); Gerhard Bouwer (Junior Analyst); Sune Sheard (Junior Analyst) Inputs and results verified by: Marrilize Bylsma (Technical Manager) Classification (DEEEP) performed by: Lizet Moore (Quality Manager)

Report authorized by: Technical Signatory

26 June 2018 Lizet Moore Marrilize Bylsma Report Date

A= Accredited NA =Not accredited O=Outsourced S=Sub-contracted NR=Not requested RTF=Results to follow The results relate only to the test item(s) tested Results marked “Not SANAS Accredited” in this report are not included in the SANAS Schedule of Accreditation for this laboratory

Results marked “Not SANAS Accredited” in this report are not included in the SANAS Schedule of Accreditation for this for Accreditation of Schedule SANAS in the included are not thisin report Accredited” SANAS “Not marked Results A= Accredited A=Accredited laboratory Table 2 dates. Table 1: END REPORTOF Literature4. references Results3. and discussion Methodology2. Analyses1. requestedand sample description included in the scope of accreditation. Any part of this report shall not be reproduced, except in full, without the the without in full, except be reproduced, not report shall this of Any part accreditation. of scope the in included 3.1 2018 3.1 2.3 Toxicitytestresults classification system Bio2.2 Sampling2.1 and sample handling

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Page 2 of 8 GCS-C-18_TOX Results marked “Not SANAS Accredited” in this report are not included in the SANAS Schedule of Accreditation for this for Accreditation of Schedule SANAS in the included are not thisin report Accredited” SANAS “Not marked Results A= Accredited A=Accredited laboratory (micro pur this For on sites/samples. based classification hazard and testing toxicity internationally Standard, way represent no in does and integrity the aquaticbiotic of in terms The environment. aquatic receiving the in biota similar towards risk/hazard the represents is based important battery to test on of that hazard note classification selected the the standardised A bodies. determined is categorywater risk receiving the of integrity biota/biological the to waters such of risk potential the determine to order in short Acute (and 2.2 Bio samples. on available are documents These request SOP06). & (SOP05 06 & 05 procedures Operating Standard Technical to Refer handling sample and Sampling 2.1 2. Methodology Table sample description and Analyses requested 1. Definitive = Series of sample dilutions tested to enhance classification accuracy dilution safe to classification enhance and tested to determine dilutions sample of = Series Definitive only tested = sample Screening 100% (undiluted) Key: included in the scope of accreditation. Any part of this report shall not be reproduced, except in full, without the the without in full, except be reproduced, not report shall this of Any part accreditation. of scope the in included DAM(Kamuzu 2)(N) MMD1 KAN MMD1 MMD2 KO MMD2 - MMDR MML5 MML4 MML3 MML2 MML1 1 algae) - . : toxicity assessments assessments toxicity

Sample name Analyses requested and description for the different samples, including sampling and delivery delivery and sampling including samples, different the for description and requested Analyses . Permission was Permission The results relate only to the test the to relate only results The

and and NA =Not =Not NA -

chronic) toxicity testing (as applied for this assessment) is applied by exposing biota to water sources water to biota byexposing is applied this assessment) for applied (as testing toxicity chronic) Poecilia reticulata Poecilia 2018/02/16 2018/02/16 2018/02/16 2018/02/16 2018/02/14 2018/02/15 2018/02/14 2018/02/17 2018/02/17

Sampling date accredited O=Outsourced S=Sub O=Outsourced accredited accepted methods and materials were applied in order to conduct acute and short and acute conduct to order in applied were materials and methods accepted granted by GCS to continue with testing despite the dela the despite testing with continue to GCS by granted pose pose GCS Water & Environment & GCSWater Environment & GCSWater Environment & GCSWater Environment & GCSWater Environment & GCSWater Environment & GCSWater Environment & GCSWater Environment & GCSWater Environment & GCSWater based on the percentage of mortalities (or inhibition (or mortalities of percentage the on based

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(fish) (fish)

were used as test organisms prior to hazard classification. prior tohazard classification. organisms test as used were 2018/06/11 2018/06/11 2018/06/11 2018/06/11 2018/06/11 2018/06/11 2018/06/11 2018/06/11 2018/06/11

item(s) tested. item(s)

Delivery date (bacteria), (bacteria),

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rpi lvl ( levels trophic Daphnia magna Daphnia

- Delivered by contracted NR=Not requested RTF=Results to follow to RTF=Results requested NR=Not contracted Any opinions/interpretations noted in this report are not are not thisin report noted Any opinions/interpretations toxicology Additional comments (sample comments Additional description or deviations) 4 SampledFebruaryin SampledFebruaryin SampledFebruaryin SampledFebruaryin SampledFebruaryin SampledFebruaryin SampledFebruaryin SampledFebruaryin SampledFebruaryin

taxonomic groups) at each of the selected selected the of each at groups) taxonomic

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Page 3 of 8 GCS-C-18_TOX Results marked “Not SANAS Accredited” in this report are not included in the SANAS Schedule of Accreditation for this for Accreditation of Schedule SANAS in the included are not thisin report Accredited” SANAS “Not marked Results A= Accredited A=Accredited laboratory test): of rate (validity mortality/immobility Control a and 2.05 of factor coverage a at 17.61% thisis test for uncertainty percentage The Ephippia numbers: Batch spread sheet EXCEL points, data relevant Karbe Spearman Trimmed used: method Statistical test endpoint:Screening Test temperature: 21 Test 4 per sample: wells of number Replicate well:5 per organisms test of Number Test 48h 24 and period: Exposure old 24h than Less age: species Test species: Test standard method: from Deviation 6341:2015 SANS method: Standard 2.2.3 test: of Validity 95% of confidence of a level and 2.36 of factor coverage a at 15.21% thisis test for uncertainty percentage The method Statistical values endpoint: Test spectrophotometer 6300 Jenway equipment: Measurement temperature: 21 Test 3 replicates: of Number A number: batch Bead MD number: batch Matrix dissolving SC number: batch Algae long cell type:10 chamber cm Test 25 mℓ volume: sample Test 72h period: Exposure species: Test None standard method: from Deviation 8692:2015 SANS method: Standard 2.2.2 test): of (validity factor Correction 95% of confidence of a level and 2.36 of factor coverage a at 11.07% thisis test for uncertainty percentage The VF dates: numbers/expiry Batch Statistical method used: Biotox software (from supplier) values endpoint: Test Measurement 2 replicates: of Number 500 µℓ volume: sample Test minutes 30 15 and period: Exposure species: Test None standard method: from Deviation 11348 SANS method: Standard 2.2.1 methods: standardized internationally usingfollowing the rooms controlled environmental in conducted were All tests included in the scope of accreditation. Any part of this report shall not be reproduced, except in full, without the the without in full, except be reproduced, not report shall this of Any part accreditation. of scope the in included

sample volume: 25 mℓ volume: sample

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Page 4 of 8 GCS-C-18_TOX Results marked “Not SANAS Accredited” in this report are not included in the SANAS Schedule of Accreditation for this for Accreditation of Schedule SANAS in the included are not thisin report Accredited” SANAS “Not marked Results A= Accredited A=Accredited laboratory • • • request: on available made be would information assurance quality following The assurance Quality buffers: 1413µS/cm of numbers Batch 95% of confidence of level 3. is test this for uncertainty percentage The multimeter used: HQ440D Instrument temperature: 25°C±3°C Test 06 Method Biotox 2.2.6 buffers: pH4 of numbers Batch 95% of confidence 2of level and a of factor thisis test for uncertainty percentage The multimeter used: HQ440D Instrument temperature: 25°C±3°C Test M Biotox 2.2.5 validation: Test 28.28% thisis test for uncertainty percentage The medium Control numbers: Batch spread sheet EXCEL points, data relevant (TSK) Karber Spearman Trimmed used: method Statistical test endpoint:Screening Test temperature: 21 Test 2 per sample: beakers number Replicate 6 per beaker: organisms test of Number mℓ 200 volume: sample Test 96h period: Exposure Test species: Test None standard method: from Deviation 203 guide OECD method: Standard 2.2.4

included in the scope of accreditation. Any part of this report shall not be reproduced, except in full, without the the without in full, except be reproduced, not report shall this of Any part accreditation. of scope the in included

species age: Less than 21 days 21 than Less age:species Participation in proficiency testing scheme (SABS, NLA) scheme testing proficiency in Participation data. and rawtest numbers batch lot, Additional In conductivity Electrical pH reticulata Poecilia ethod 05 05 ethod . - house The results relate only to the test the to relate only results The

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10% effect 10% is regardedeffect as tests performed, the performed, tests tures based on a set of and plant seed growth)

Page 6 of 8 GCS-C-18_TOX Results marked “Not SANAS Accredited” in this report are not included in the SANAS Schedule of Accreditation for this for Accreditation of Schedule SANAS in the included are not thisin report Accredited” SANAS “Not marked Results A= Accredited A=Accredited laboratory II)based the on 32%and 26%b hazard showed DAM and MMDR MML5, MML4, MML3, MML2, MML1, Samples Table samples. the different of classification hazard overall and results test individual for 2 below totable Refer 3.1 201 anddiscussion 3. Results site/sample name shaded in orange definitive= test site/sample name shaded in purple screening= test Weight (%) = capricornutum *** byfiltration. affected if detected be still will toxicity hence and filtered never are samples guppytest and daphnia (F) = = S.D.O.T.H Algal= ** test result inconclusive due to interference (caused precipitate forming in the sample during testing). As the degree of inhibition/stimulation* is unknown, individual test result notwas used for overall hazard classification. % WQ included in the scope of accreditation. Any part of this report shall not be reproduced, except in full, without the the without in full, except be reproduced, not report shall this of Any part accreditation. of scope the in included = EC/LC= values not determined, definitive testing required if a hazard observedwas and overpersists subsequent sampling runs

Key: WQ = for= definitive testing, only the 100% concentration (undiluted) sample mortality/inhibition/stimulation is reflected by this table.summary The dilution series results are considered for EC/LC values and Toxicity unit determinations.

= The= overall hazard classification takes into account the full battery of andtests is not based on a single test result. Note that the overall hazard classification is expressed as acute/chronic level of toxicity, due to the fact that the P. reticulata D. magna S. capricornutum V. fischeri Water = Water = quality at the time of starting the

Inhibition/Mortality rate with "(F)" indicates that the sample filtered,was this is often essential with turbid or coloured samples to perform the algae and bacteria tests. Filtration could potentially lower the toxicity for the specific test, but (guppy) (A) (waterflea) (A) (micro-algae) (A) (bacteria) (A)

Overall classificationOverall - Hazard class*** quality Toxicity unit (TU) / Description EC/LC50 (96hours) EC/LC10 (96hours) % Test started on yy/mm/dd Toxicity unit (TU) / Description EC/LC50 (48hours) EC/LC10 (48hours) % Test started on yy/mm/dd Toxicity unit (TU) / Description EC/LC50 (72hours) EC/LC20 (72hours) % Test started on yy/mm/dd Toxicity unit (TU) / Description EC/LC50 (30 mins) EC/LC20 (30 mins) % Test started on yy/mm/dd Dissolved oxygen (mg/l) (N) EC (Electrical conductivity) @ 25°C (mS/m) (A) pH @ 25°C (A) 96hour mortality rate (-%) 48hour mortality rate (-%) 72hour inhibition (-) / stimulation (%)(+) 30min inhibition (-) / stimulation (%)(+) 2 ” 8 Some degree of acute/chronic toxic hazard based on this single test organism, refer to overall hazard classification, which takes into account the full battery of test organisms. :

(Class I). I). (Class relative toxicity levels (out of 100%), higher values indicate that more of the individual indicatedtests toxicity within a specific class

(micro-algae) and the - . 0 Test results and risk classification for water samples during during watersamples classificationfor and results risk Test 2 The results relate only to the test the to relate only results The

Weight (%) survey survey NA =Not =Not NA Results Samples MMD1 KAN and MMD2 KOMMD2 andSamples KAN MMD1 V. fischeri -

water accredited O=Outsourced S=Sub O=Outsourced accredited Daphnia magna tests aretests regarded as short-chronic levels of toxicity andtests the overall classification therefore contains a degree of chronic toxicity assessment.

no acute hazard no acute hazard no short-chronic chronic hazard IIClass - Slight MMD1 KAN MMD1 S.D.O.T.H. 18/06/18 18/06/12 18/06/13 18/06/21 hazard acterial light emission inhibition noted for thesesamples testing. 10,7 5(F) -32 6,2 6,9 25 0 0 * * * * * * * * written approval of Biotox Laboratory Services Laboratory Biotox of approval written no acute hazard no acute hazard chronic hazard IIClass - Slight S.D.O.T.H. KO MMD2 18/06/21 18/06/18 18/06/12 18/06/13 39,0 -26 6,4 7,1 33 -5 ** 0 ** ** ** * * * * * *

item(s) tested. item(s) no short-chronic no short-chronic no acute hazard no acute hazard acute/chronic Class IClass - No 18/06/18 18/06/12 18/06/13 18/06/21 hazard hazard MML1 hazard -12 7,0 6,3 7,3 -8 -5 5 0 * * * * * * * * showed a showed

no acute hazard no acute hazard no short-chronic no short-chronic - acute/chronic contracted NR=Not requested RTF=Results to follow to RTF=Results requested NR=Not contracted Any opinions/interpretations noted in this report are not are not thisin report noted Any opinions/interpretations Class IClass - No 18/06/21 18/06/18 18/06/12 18/06/13 hazard hazard MML2 hazard 26,1 7,8 7,7 12 -9 0 0 0 * * * * * * * * “ slight chronic environmental toxicity hazardchronicenvironmentalslight toxicity no acute hazard no acute hazard no short-chronic no short-chronic February acute/chronic Class IClass - No 18/06/21 18/06/18 18/06/12 18/06/13 hazard hazard MML3 hazard 10,2 . 7,5 7,6 13 -9 0 0 0 * * * * * * * * “ no acute/chronic no

no acute hazard no acute hazard no short-chronic no short-chronic 2018 acute/chronic Class IClass - No

18/06/21 18/06/18 18/06/12 18/06/13 hazard hazard MML4 hazard 10,9 -13 7,3 7,4 18 -8 0 0 * * * * * * * * .

no acute hazard no acute hazard no short-chronic no short-chronic acute/chronic respectively during testing. Class IClass - No 18/06/21 18/06/18 18/06/12 18/06/13 hazard hazard MML5 hazard 11,1 -18 7,2 7,4 10 0 0 0 * * * * * * * * environmental no acute hazard no acute hazard no short-chronic no short-chronic acute/chronic Class IClass - No 18/06/21 18/06/18 18/06/12 18/06/13 MMDR hazard hazard hazard 27,5 -10 -16 5,6 7,3 14 0 0 * * * * * * * *

no acute hazard no acute hazard no short-chronic no short-chronic acute/chronic 2) (Kamuzu Class IClass - No

18/06/21 18/06/18 18/06/12 18/06/13 hazard hazard hazard ” toxicity DAM -18 7,5 8,2 7,0 17

0 0 0 * * * * * * * * (Class

S. .

Page 7 of 8 GCS-C-18_TOX Results marked “Not SANAS Accredited” in this report are not included in the SANAS Schedule of Accreditation for this for Accreditation of Schedule SANAS in the included are not thisin report Accredited” SANAS “Not marked Results A= Accredited A=Accredited laboratory measuring for Method 1993. EPA), (US AGENCY PROTECTION ENVIRONMNETAL STATES UNITED ENVIRONMNETAL STATES UNITED TöRöKNE I, BLINOVA M, BLAHOSLAV G, PERSOONE F INC. 2012. DAPHTOXKIT MICROBIOTEST quality “Water 1998. Standard, EUROPEAN The 2003. FORESTRY, AND AFFAIRS OF WATER DEPARTMENT BO1243 2012. Oy. ABOATOX 4 . Literature references . Literature included in the scope of accreditation. Any part of this report shall not be reproduced, except in full, without the the without in full, except be reproduced, not report shall this of Any part accreditation. of scope the in included Office of Research and Development, Development, and Washington. Research of Office EPA/600/4 organisms. marine and freshwater to waters receiving and effluent of toxicity test toxicity acute Fish communication). (personal wastewaters and waters natural for Microbiotests with user L,B.practical and KOLAR A TOTHOVA L, L, STEPANOVA TOFAN Belgium. (Gent), Mariakerke 15, 9030 Kleimoer Procedure. Operational ISO 11348 of emission Pretoria. Studies, Quality of Institute Water document. discussion a approach, (DEEEP) Potential Effect Ecological of Estimation Direct the Introducing Discharges. www.aboatox.com . The results relate only to the test the to relate only results The NA =Not =Not NA - 3. European Committee for Standardization, Brussels. Standardization, for Committee European 3. Vibriofischeri accredited O=Outsourced S=Sub O=Outsourced accredited

–

- Freshwater and marine. OPPTS 850.1075. Report number EPA number Report 850.1075. OPPTS marine. and Freshwater 0 BooT Kt Isrcin fr s. aiua . FIN 2. Savikuja use. for Instructions Kit. BioToxTM 500 written approval of Biotox Laboratory Services Laboratory Biotox of approval written (Luminescent bacteria test) test) bacteria (Luminescent PROTECTION AGENCY (US EPA), 1996. Ecological effects test guidelines. test effects Ecological 1996. EPA), (US AGENCY PROTECTION

item(s) tested. item(s) TM ENDOFREPORT –

MAGNA. Crustacean Toxicity Screening Test for freshwater. Standard for freshwater. Screening Test Toxicity MAGNA. Crustacean

D etermination of the inhibitory effect of water samples on the light light the on samples water of effect inhibitory the of etermination

A, ZARINA T, MANUSADZIANAS L, NALECZ L, MANUSADZIANAS T, ZARINA A,

Part 3 for the method using freeze using method the for 3 Part Management of Complex Industrial Waste Water Water Industrial Waste Complex of Management

www.microbiotest.be

- friendly toxicity classification system system toxicityfriendly classification

- 21250, Masku Finland. Finland. Masku 21250, - - 90/027F, 4 90/027F, 712 - dried bacteria”, EN EN bacteria”, dried .

- c - - 96 JAWECKI G, G, JAWECKI -

118. the acute acute the th

edition. edition.

Page 8 of 8 GCS-C-18_TOX