Appendix C

Toxicity of Malathion to Marine and Estuarine Fish and Invertebrates

This appendix presents results of laboratory toxicity studies that yielded acute and chronic toxicity endpoints for the effects of malathion on marine/estuarine fish, , and mollusks. These studies include ones submitted by pesticide registrants for fulfillment of FIFRA testing requirements for pesticide registration as well as comparable studies that have been published in the open literature. Open literature studies are listed when they yielded similar endpoints as a required EPA guideline study, namely an LC50 or EC50 for acute exposure, or an NOAEC and LOAEC for growth, development, or reproduction for chronic studies. We are not attempting to summarize the complete body of toxicity literature on effects to marine and estuarine species.

Relevant studies from the open literature were identified from a screen of data in the EPA’s ECOTOX database with toxicity results on malathion. In order to be included in the ECOTOX database, papers must meet the following minimum criteria: 1. The toxic effects are related to single chemical exposure; 2. The toxic effects are on an aquatic or terrestrial plant or species; 3. There is a biological effect on live, whole organisms; 4. A concurrent environmental chemical concentration/dose or application rate is reported; and 5. There is an explicit duration of exposure.

Data that pass the ECOTOX screen are further evaluated for use in the assessment along with the registrant-submitted data, and may be incorporated qualitatively or quantitatively into this endangered species assessment.

Acute Toxicity to Fish

Acute toxicity testing with estuarine/marine fish species using the TGAI is required for malathion because the end-use product is intended for direct application to the marine/estuarine environment and the active ingredient is expected to reach this environment because of its use near estuarine environments. The preferred estuarine test species is sheepshead minnow. Results of acute tests with marine/estuarine fish that were submitted for FIFRA testing requirements and reviewed by the EPA are summarized in Table C1. Table C2 provides additional information on the acute toxicity of malathion to marine and estuarine fish from published studies identified in the ECOTOX database.

1- Table C1. Acute Toxicity of Malathion to Marine/Estuarine Fish % LC50 (95% CI) Classi- Species Tested ai Duration in µg/L MRID Author fication

48 hr Spot(SW) 95 LC50=320 (N.R.) 40228401 F. L. Mayer, USEPA Supl.

48 hr Striped mullet(SW) 95 LC50=330 (N.R.) 40228401 F. L. Mayer, USEPA Supl.

48 hr Longnose 95 LC50=150 (N.R.) 40228401 F.L. Mayer, USEPA Supl. killifish(SW)

96 hr Sheepshead 95 LC50=33.0 (14-63) 41174301 Bowman, J 1989, ABC Accept. minnow(SW) Laboratories

96 hr Striped bass(SW) 95 LC50=60 (N.R.) 156311 Wellborn, T. 1971 Supl. Reference

96 hr Sheepshead 57 LC50 55 41252101 Bowman,J. ABC Labs, Accept. minnow(SW) EC 1989

Table C2. Data from the open literature on the acute toxicity of malathion to saltwater fishes. ECOTOX Results Ref # Common Name Scientific Name Duration Endpoint (µg/L)

522 Threespine stickleback Gasterosteus aculeatus 96 hr LC50 94

522 Threespine stickleback Gasterosteus aculeatus 96 hr LC50 76.9

628 American eel Anguilla rostrata 96 hr LC50 82

628 Atlantic silverside Menidia menidia 96 hr LC50 125

628 Bluehead Thalassoma bifasciatum 96 hr LC50 27

628 Mummichog Fundulus heteroclitus 96 h LC50 80

628 Mummichog Fundulus heteroclitus 96 h LC50 400

628 Northern puffer Sphoeroides maculatus 96 h LC50 3250

628 Striped killifish Fundulus majalis 96 h LC50 250

628 Striped mullet Mugil cephalus 96 h LC50 550

5074 Sheepshead minnow Cyprinodon variegatus 96 h LC50 51 Lepidocephalichthys 5629 Loach thermalis 48 h LC50 7750 Lepidocephalichthys 5629 Loach thermalis 48 h LC50 20610 Lepidocephalichthys 5629 Loach thermalis 24 h LC50 22690 100% 81672 Red drum Sciaenops ocellatus 24 h mortality 1960 0% 88204 Gilthead seabream Sparus aurata 96 h mortality 386.8

2- Chronic Toxicity To Freshwater and Estuarine Fish

A literature search conducted with the ECOTOX database yielded only one study that provided information on the chronic toxicity of malathion to an marine/estuarine fish that yielded results comparable to the EPA test guideline. This study was an unpublished Ph.D. dissertation that reported acute and chronic toxicity of malathion to the red drum (Sciaenops ocellatus) larvae (ECOTOX Ref. No. 081672, Alvarez, 2005). According to this report, malathion did not cause any significant effects on the growth rate, behavior, or respiration rates in either of the two test concentrations (1.0 and 10 µg/L, nominal). The measured day-0 concentration of the higher test concentration was 7.4 µg/L. The study therefore established an NOAEC for the Red drum at 7.4 µg/L, but an LOAEC was not determined. The Agency will review the study report and raw data during the registration review process to determine whether these results are acceptable for use in the EPA risk assessment.

Toxicity of Malathion Formulations to Fish

A single study submitted by the registrant in 1989 with sheepshead minnow exposed to 57% EC formulation showed this product to be highly toxic with an LC50 of 33 ppb (MRID 41252101).

Acute Toxicity to Marine/Estuarine Invertebrates

Acute toxicity testing with estuarine/marine invertebrates using the TGAI was required for malathion because the end-use product is intended for direct application to the marine/estuarine environment or the active ingredient is expected to reach this environment because of its use near estuarine habitats. Testing is required with both a and a mollusk. The preferred test species are the mysid and the eastern oyster. Results of acute tests with marine/estuarine fish that were submitted for FIFRA testing requirements and reviewed by the EPA are summarized in Table C3. Table C4 provides additional information on the acute toxicity of malathion to marine and estuarine fish from published studies identified in the ECOTOX database.

Table C3. Acute Toxicity of Malathion to Marine/Estuarine Crustaceans

Species Tested % Durati EC50 or LC50 MRID Author Classifi- ai on (95% CI) in µg/L cation

96 hr Mysid, 94 LC50=2.2 (1.5-2.6) 41474501 Forbis, A., ABC Accept. bahia Lab.,1990

48 hr Pink , 95 48 Hr LC50=280 40228401 F.L. Mayer, USEPA, Suppl. Penaeus duorarum (N.R.) 1986

96 hr Eastern oyster, 95 96 Hr LC50>1000 40228401 F.L. Mayer, USEPA, Suppl. Crassostrea 1986 virginica

96 hr Eastern oyster, 57% 96 Hr EC50=2960 42249901 Wade, B and J. Wisk, Accept.

3- Crassostrea EC (N.R.) ESE , Inc. 1992 virginica

48 hr Blue Crab, 95 48 Hr LC50>1000 40228401 F.L. Mayer, USEPA, Suppl. Callinectes sapidus 1986

Table C4. Data from the open literature on the toxicity of malathion to saltwater crustaceans. ECOTOX Results Ref # Common Name Scientific Name Duration Endpoint (µg/L)

968 Opossum shrimp bahia 96 h LC50 3.0

968 Opossum shrimp Americamysis bahia 96 h LC50 3.1

968 Opossum shrimp Americamysis bahia 96 h LC50 2.6

2280 Opossum shrimp Americamysis bahia 96 h LC50 5.7

2280 Opossum shrimp Americamysis bahia 96 h LC50 3.2

2280 Opossum shrimp Americamysis bahia 96 h LC50 5.4

2280 Opossum shrimp Americamysis bahia 96 h LC50 5.2

2280 Opossum shrimp Americamysis bahia 96 h LC50 4.0

2280 Opossum shrimp Americamysis bahia 96 h LC50 5.0 Dungeness or edible 100% 6793 crab Cancer magister 6 d mortality 2.0 Dungeness or edible 6793 crab Cancer magister 96 h EC50 0.40 Dungeness or edible 6793 crab Cancer magister 96 h LC50 1300 Dungeness or edible 6793 crab Cancer magister 96 h LC50 1.2 Dungeness or edible 100% 6793 crab Cancer magister 12 d mortality 2400

13513 Northern pink shrimp Penaeus duorarum 96 h LC50 12

13513 Opossum shrimp Americamysis bahia 96 h LC50 11 Daggerblade grass 14346 shrimp Palaemonetes pugio 96 h LC50 9.1 Daggerblade grass 14346 shrimp Palaemonetes pugio 96 h LC50 38 Daggerblade grass 14346 shrimp Palaemonetes pugio 96 h LC50 13 Daggerblade grass 14346 shrimp Palaemonetes pugio 18 d LC50 13 Daggerblade grass 14346 shrimp Palaemonetes pugio 25 d LC50 6.5 Daggerblade grass 14346 shrimp Palaemonetes pugio 35 d LC50 5.6

4- ECOTOX Results Ref # Common Name Scientific Name Duration Endpoint (µg/L)

16752 Whiteleg shrimp Penaeus vannamei 96 h LC50 21

18363 Brine shrimp Artemia sp. 24 h EC50 > 140,000

19281 Harpacticoid Tigriopus brevicornis 96 h LC50 21

19281 Harpacticoid copepod Tigriopus brevicornis 96 h LC50 24

19281 Harpacticoid copepod Tigriopus brevicornis 96 h LC50 7.2

51439 Gammarid amphipod Gammarus palustris 72 h LC50 5.9

51439 Gammarid amphipod Gammarus palustris 72 h LC50 19

51439 Gammarid amphipod Gammarus palustris 96 h LC50 2.3

51439 Gammarid amphipod Gammarus palustris 96 h LC50 4.7

70574 Brine shrimp 24 h LC50 37,000

73331 American lobster Homarus americanus 96 h LC50 38 Metapenaeus 89575 Sand shrimp monoceros 96 h LC50 1400

Additional data on the toxicity of malathion to the blue crab Callinectes sapidus was recently reported in a master’s thesis by Christina Wendel (Wendel, 2008). Effects of malathion exposure on the mortality of juvenile and adult crabs were evaluated using time-to-death analysis. Results from this study are presented in Table C5. While not directly comparable to results from LC50 studies, these results provide useful supplemental information on the lethality of malathion to crustaceans.

Table C5. Results from Wendel (2008) on the toxicity of malathion to the blue crab. Results Common Name Scientific Name Age Endpoint (µg/L) Mortality, time- NOAEC = 0.32 Blue crab Callinectes sapidus Juvenile to-death LOAEC = 1.0 Mortality, time- NOAEC = 1.0 Sand shrimp Callinectes sapidus Adult to-death LOAEC = 11

Several toxicity studies are also available on the effects of malathion to estuarine/marine mollusks. Table C6 provides results of acute toxicity tests with estuarine/marine mollusks based on studies submitted by pesticide registrants to fulfill EPA test requirements. Table C7 provides additional information on the acute toxicity of malathion to marine/estuarine mollusks from published studies identified in the ECOTOX database.

5- Table C6. Acute Toxicity of Malathion to Marine/Estuarine Mollusks

Species Tested % Duration EC50 or LC50 MRID Author Classifi- ai (95% CI) in µg/L cation

96 hr Eastern oyster, 95 96 Hr LC50>1000 40228401 F.L. Mayer, USEPA, Suppl. Crassostrea 1986 virginica (SW)

96 hr Eastern oyster, 57 96 Hr EC50=2960 42249901 Wade, B and J. Wisk, Accept. Crassostrea % (N.R.) ESE , Inc. 1992 virginica(SW) EC

Table C7. Data from the open literature on the toxicity of malathion to saltwater mollusks. ECOTOX Results Ref # Common Name Scientific Name Duration Endpoint (µg/L)

7917 Salt marsh snail Melampus bidentatus 48 h LC50 100,000

8127 Blue mussel Mytilus edulis 96 h LC0 39,000

8127 Blue mussel Mytilus edulis 96 h LC100 6900

14269 Marine bivalve Katelysia opima 96 h LC50 6.0

14269 Marine bivalve Katelysia opima 96 h LC0 1.0 Northern quahog or Mercenaria 0% 17307 hard clam mercenaria 96 h mortality 37,000 American or virginia 2400 oyster Crassostrea virginica 14 d LC50 2700

Sublethal Toxicity to Freshwater and Marine Invertebrates

A literature search conducted with the ECOTOX database yielded no study that provided endpoints on the chronic toxicity of malathion to an marine/estuarine invertebrates that are comparable to endpoints that would be obtained from an EPA guideline study. One study with results somewhat similar to that of the chronic mollusk guideline was a study on embryonic effects of malathion exposure to the Mediterranean mussel, Mytilus galloprovincialis (ECOTOX Ref. No. 817421, Losso et al, 2004). According to this report, the 48-hr EC50 for abnormal development was 2200 µg/L. This study differs from the guideline study for chronic mollusk toxicity in that it involved an acute (48-hr) exposure period and did not evaluate the entire live cycle of the organism. The Agency will review this report during the registration review process and determine if and how the results might be used in the risk assessment of sublethal effects to saltwater invertebrates.

References

Table C8 provides citations of studies that were identified in the ECOTOX literature

6- search and identified in this appendix by Eco Ref. No.

Table C8. Citations of references that were cited by ECOTOX Ref. No. Ref . Citation Katz M; 1961. Acute Toxicity of some Organic Insecticides to Three Species of Salmonids and to the 522 Threespine Stickleback. Trans Am Fish Soc 90(3): 264-268 Eisler R; 1970. Acute Toxicities of Organochlorine and Organophosphorus Insecticides to Estuarine Fishes. 628 Tech Pap No 46, Bur Sport Fish Wildl , U S D I , Washington, DC. Goodman LR, GM Cripe, PH Moody, and DG Halsell. 1988. Acute Toxicity of Malathion, Tetrabromobisphenol-A, and Tributyltin Chloride to Mysids (Mysidopsis bahia) of Three Ages. Bull Environ 968 Contam Toxicol 41(5): 746-753 Cripe GM, A Ingley-Guezou, LR Goodman, and J Forester. 1989. Effect of Food Availability on the Acute Toxicity of Four Chemicals to Mysidopsis bahia (Mysidacea) in Static Exposures. Environ Toxicol Chem 2280 8(4): 333-338 Davis HC, H Hidu. 1969. Effects of Pesticides on Embryonic Development of Clams and Oysters and on 2400 Survival and Growth of the Larvae. Fish Bull 67(2): 393-404 Tagatz ME, PW Borthwick, GH Cook, and DL Coppage. 1974. Effects of Ground Applications of Malathion 2895 on Salt-Marsh Environments in Northwestern Florida. Mosq News34(3/4): 309-315 Hansen DJ and PR Parrish. 1977. Suitability of Sheepshead Minnows (Cyprinodon variegatus) for Life- Cycle Toxicity Tests. In: F L Mayer and J L Hamelink (Eds ), and Hazard Evaluation, 1st 5074 Symposium, AST Kumari SDR and NB Nair. 1978. Toxicity of Some Insecticides to Lepidocephalus thermalis (Cuv. & Val.). 5629 Proc Indian Natl Sci Acad 44(3): 122-132 Caldwell RS; 1977. Biological Effects of Pesticides on the Dungeness Crab. "EPA-600/3-77-131, U S EPA, 6793 Gulf Breeze, FL(): 143 p.(U.S.NTIS PB-276978)-" Fitzpatrick G; 1975. Impact of Temefos and Other Mosquito Larvicides on the Salt Marsh Snail Melampus bidentatus Say (Basommatophora: Ellobiidae). "Ph D Thesis, Rutgers University, Rutgers, NJ:120 p ; Diss 7917 Abstr Int B Sci Eng 36(5): 2045-2046 (Pub Liu DHW;Lee JM; 1975. Toxicity of Selected Pesticides to the Bay Mussel (Mytilus edulis). "EPA-660/3-75- 8127 016, U S EPA, Corvallis, OR(): 102 p. (U.S.NTIS PB-243221)-" Cripe GM; 1994. Comparative Acute Toxicities of Several Pesticides and Metals to Mysidopsis bahia and 13513 Postlarval Penaeus duorarum. Environ Toxicol Chem 13(11): 1867-1872 Akarte SR;Muley DV;Mane UH; 1986. "Effect of Cythion-Malathion 50 EC (W/W) on the Estuarine Clam, Katelysia opima (Gmelin)". "In: M F Thompson, R Sarojini, and R Nagabhushanam (Eds ), Biology of 14269 Benthic Marine Organisms: Tech Key PB;Fulton MH;Scott GI;Layman SL;Wirth EF; 1998. "Lethal and Sublethal Effects of Malathion on 14346 Three Life Stages of the Grass Shrimp, Palaemonetes pugio". Aquat Toxicol 40(4): 311-322 Salame MJ;Rouse DB;Yeh HS; 1996. Environmental Toxicity of Malathion to the Marine Shrimp Penaeus vannamei. "Manuscript, Inacua S A , Investigacion y Acuacultura, Ecuador; Dep Fisheries and Allied 16752 Aquaculture Eisler R;Weinstein MP; 1967. "Changes in Metal Composition of the Quahaug Clam, Mercenaria mercenaria, 17307 After Exposure to Insecticides". Chesapeake Sci 8(4): 253-258 Guzzella L;Gronda A;Colombo L; 1997. Acute Toxicity of Organophosphorus Insecticides to Marine 18363 Invertebrates. Bull Environ Contam Toxicol 59(2): 313-320 Forget J;Pavillon JF;Menasria MR;Bocquene G; 1998. "Mortality and LC50 Values for Several Stages of the Marine Copepod Tigriopus brevicornis (Muller) Exposed to the Metals Arsenic and Cadmium and the 19281 Pesticides Atrazine, Carbofuran, Dichlorvos, and Ma.

7- Leight AK;Van Dolah RF; 1999. "Acute Toxicity of the Insecticides Endosulfan, Chlorpyrifos, and Malathion to the Epibenthic Estuarine Amphipod Gammarus palustris (Bousfield)". Environ Toxicol Chem 18(5): 958- 51439 964 Sujatha CH;Nair SM;Chacko J; 1995. Pesticide Induced Physiological Changes in an Estuarine Clam. 60150 Oebalia21(): 181-186

69472 Shacklock PF;Croft GB; 1981. Effect of Grazers on Chondrus crispus in Culture. Aquaculture22(): 331-342 Wilkins RM;Metcalfe RJ; 1993. Toxicity of Soil Applied Herbicides to Brine Shrimp Larvae (Artemia salina) 70574 and Synergism with Other Pesticides. Br Crop Prot Conf - Weeds1(): 163-168 De Guise S;Maratea J;Perkins C; 2004. Malathion Immunotoxicity in the American Lobster (Homarus 73331 americanus) upon Experimental Exposure. Aquat Toxicol 66(4): 419-425 Alvarez MC; 2005. "Significance of Environmentally Realistic Levels of Selected Contaminants to Ecological Performance of Fish Larvae: Effects of Atrazine, Malathion, and Methylmercury". "Ph D Thesis, Univ of 81672 Texas, Austin, TX(): 141 p.-" Losso C;His E;Ghetti PF;Ghirardini AV; 2004. Sensitivity of Embryotoxicity Test with Mytilus galloprovincialis (LMK) Towards Some Compounds of Environmental Interest (Copper and Pesticides). 81741 Environ Technol 25(7): 841-846 Rosety M;Rosety-Rodriguez M;Ordonez FJ;Rosety I; 2005. Time Course Variations of Antioxidant Enzyme Activities and Histopathology of Gilthead Seabream Gills Exposed to Malathion. Histol Histopathol 20(4): 88204 1017-1020

Nair GA;Nair NB; 1982. "Effect of Certain Organophosphate Biocides on the Juveniles of the Isopod, 89498 Alitropus typus M. Edwards (Crustacea: Flabellifera: Aegidae)". J Anim Morphol Physiol 29(1/2): 265-271

Reddy MS;Jayaprada P;Rao KVR; 1990. "Impact of Methylparathion and Malathion on Cholinergic and Non- 89575 Cholinergic Enzyme Systems of Penaeid Prawn, Metapenaeus monoceros". Biochem Int 22(4): 769-780

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