Appendix 2 Open Literature Review Summaries for Malathion
Description of Use in Document
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Description of Use in Document: Valid for arrays (qualitative) Rationale for Use: Based on the limitations described below. Limitations of Study: Main reasons: Variability around the mean endpoint values are not reported. While the study authors state that survival and reproduction in the treatment groups were significantly different than the control, there is uncertainty in the time component of this. In Figure 2, the study authors state that differences between the treatments and control are indicated by closed circles for which each measured value at each time point for all treatments are closed circles. In looking at the figure, while there does appear to be a visual decrease in mean survival and reproduction at the test concentration compared to the controls over time (variability around the mean value not provided), it appears that at least for some of the days (at beginning of study), the treatments do not appear to be different from the control. The lack of understanding variability around the mean value adds to this uncertainty. Also for survival, control survival was approximately 20% by day 8 which is far below recommended (OCSPP 850 and ASTM) control survival rates. For the acute mortality rates, definitive LC50 values were not reported, only the range in which the LC50 was estimated to be between. Other limitations: Water chemistry parameters were not reported. Test concentrations were not measured. Xylene was added as a treatment group. There is uncertainty in whether this was added due to xylene being present in the formulation or xylene was used to solubilize the test material (a formulation with unknown impurity profile relative to current standards). It is also unclear whether this treatment group was added for both the acute and chronic or only chronic. Mortality rates were not reported for the control group in the acute study. Primary Reviewer: Amy Blankinship, ERB6 Secondary Reviewer Elizabeth Donovan, ERB6 Chemical Name: Malathion PC Code: 057701 ECOTOX Record Citation: Cothran, R. D., R.L. Greco and R.A. Relyea. 2009. Environ. Toxicol. 25(3): 310-314. No Evidence that a Common Pesticide Impairs Female Mate Choice in a Freshwater Amphipod. E120900 Purpose of Review: Endangered Species Assessment Date of Review: March 1, 2015 Summary of Study Findings: Methods Hyalella spp. were field collected (from Lake Le Boueuf, Erie County PA near shore habitats to try to ensure the same species (B clade spp. collected)) and maintained in 90L wading pools with a 60% shade cloth. Studies conducted indoors at 22.7±0.9°C on a 12:12 light cycle, and amphipods were not fed during acute mortality studies. Seven malathion treatments each with five replicates (99.1%, Chem Service Lot No. 396-71B) were used for each study. Three different amphipod classes were evaluated: females, small males and large males. Well water and solvent control (1% ethanol) were also included, and test solutions were renewed daily (96 hr exposure). Several test concentrations were measured: nominal concentrations of 10.0, 5.0, 1.0, 0.05, and 0.01 µg/L; actual concentrations 10.4, 1.52, 0.16, 0.05, and none detected (ND; LOD 0.05 µg/L) µg/L. Petri dishes (100 mm x 20mm) were filled with 40mL of test solution and a Nitex screen (2cm2) was added for substrate. Ten organisms were added to each dish. Males were visually assigned to size class and confirmed based on measurement of head length on a subset of organisms. Mortality was recorded daily. 96-hr LC50 values were calculated using probit regression analysis. Additionally, repeated measures ANOVA and Tukey tests were used to test for effect of malathion concentration on survival. The authors stated that the goal of the acute mortality test was to derive sublethal concentrations to examine potential effects on female mate choice. In the sublethal test, experimental units were the same as the acute study except that the amphipods were fed during the study (Tetramin and spirulina). Pairing patterns (females with either small or large males) were observed for three malathion concentrations (0.55, 0.38 and 0.05 µg/L) four times daily. There were 20 replicates per treatment level. A replicate was excluded if a pairing was missed (presence of embryos; 25% of cases) or an individual died (28% of cases). Pearson X2 test used to evaluate pairing patterns and malathion treatments. Results There was no significant differential sensitivity in acute exposure among the groups. Control mortality was 6.22-12.22% in females, 2-20% in small males and 34-38% in large males. The 96-hr LC50 values are reported below (study contained figures for each test group presenting cumulative mortality at 24 hr intervals). Given the higher mortality rate in the large males, the study authors emphasized caution in the large male toxicity values. There were no effects reported for female mate choice.
Description of Use in Document: Valid for arrays (qualitative) for acute LC50 values for females and small males. Invalid for large males. Rationale for Use: Based on the limitations described below (including test material information). Limitations of Study: Main reasons: Control mortality for large males was high (34-38%) and therefore, the acute LC50 values were not considered reliable. Other limitations: Animals and vegetation were field collected, therefore, prior exposure history to potential contaminants is unknown. Additionally, it was acknowledged that different species of Hyalella may have been present in the assays (although measures to limit this were reported during collection). In the female mate choice, examination of potential influence of different species was reported and no impact was observed. The lowest treatment group in the acute studies was below the limit of detection and since the measured concentration in the other treatments were not always at/near nominal, there is uncertainty in what the actual concentration was in this treatment as well as how it was reported in the LC50 calculations. While the source and lot# of the test material was reported, it is unknown from this study the impurity profile relative to current standards. Primary Reviewer: Amy Blankinship, ERB6 Secondary Reviewer Elizabeth Donovan, ERB6 Chemical Name: Malathion CAS NO: 121-75-5 ECOTOX Record Number and Citation: 103059 McCarthy ID and Fuiman LA. 2008. Growth and Protein Metabolism in Red Drum (Sciaenops ocellatus) Larvae Exposed to Environmental Levels of Atrazine and Malathion. Aquat. Toxicol. 88: 220-229 Purpose of Review: Endangered Species Assessment Date of Assessment: 2/19/15 Brief Summary of Study Findings: Methods Red drum larvae (Sciaenops ocellatus; 7-8 mm length and 17-21 days post-hatch) from laboratory cultures were exposed to malathion (98%) and atrazine (98%), both sourced from Chem Service Inc., which was dissolved in acetone. The solvent concentration did not exceed 10 µL/L in the exposure tanks (50L fiberglass tanks). Two replicates were used for each treatment group: 40 and 80 µg/L for atrazine and 1 and 10 µg/L for malathion; a solvent control was also used. Test solution samples were collected 5 minutes and 96 hours after exposure. 500 larvae were used in each experiment. Based on the study, it appears that the assay was repeated four times for each chemical using a different spawn for each assay. Results are presented as means across the repeat studies (spawns). Distilled water was added daily to account for evaporation (renewal of test solutions was not reported), and water temperature and salinity were maintained at 26.1±0.1 ◦C and 28.9±0.1PSU. Larvae were fed daily brine shrimp (Artemia salina) nauplii and maintained on a 12:12 L:D cycle. For each spawn/replicate, total length, wet weight and protein content were measured on days 0, 1, 2, 4 and 8. Protein synthesis measurements were conducted on day 2, 4, and 8. An average value was calculated for the duplicate tanks within each treatment. For growth 20 larvae were sampled from the culture on day 0 and from each treatment tank on days 1, 2, 4 and 8 approximately 24 hours after feeding. Total length was measured photographically. Protein synthesis was measured using the flooding dose method of Garlick et al 1980, modified by Houlihan et al 1995. The analysis was conducted using a sample of fish weighing 100 mg (wet weight) and the number of fish necessary (to reach 100 mg) was calculated using a set of equations based on total length. Growth rates were analyzed using analysis of covariance and protein synthesis rates were analyzed using two-way analysis of variance. Post-hoc pair-wise comparisons were conducted using least squares difference test. Results Measured test concentrations are presented in Table 1. Neither atrazine nor malathion was detected in the control. It was also reported that an average of 17±10% atrazine had degraded to desethyl-atrazine after 4 days. No other degradation products were detected.
Atrazine
There were no significant differences in growth rate based on length. The study author reported depressions in growth rates based on wet weight (p=0.05) and protein content (p=0.06), with a significant decreasing trend in growth rate for wet weight (p=0.01) (trend for protein content p value was 0.08). Data is presented in Figure 1 (based on Figure 1 in paper). Rates of protein synthesis were significantly increased at 40 µg/L on day 4 and 8 and at 80 µg/L on day 8 (shown as Table 2 below). 
Malathion There was no significant difference in growth rate based on length or protein content. The study author reported a significant depression in average growth rate based on wet weight (p=0.03) in fish exposed to malathion. While not significant, the trend for growth rates based on protein content was similar to wet weight. Data is presented in Figure 2 below. Rates of protein synthesis were reported to be significantly increased at 10 µg/L on day 2 only (Table 2 below did not indicate significance for this day, treatment, review assumes it was an oversight). 
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