Description of Use in Document

1. 
   [Major] There is uncertainty in whether an approximate 33-50% increase in plaque formation (increase appears to be consistent over a 10,000 fold range in test dose), respectively, is sufficient to result in an adverse effect on reproduction or survival, as there were no effects on other immune responses, AChE, weight, water or food consumption, or signs of toxicity.
   
2. 
   The study was conducted with a formulation purchased in Canada. There is uncertainty in if the formulation and its impurities is representative of current standards in the United States.
   

Fujimura, R., B. Finlayson, and G. Chapman. 1991. Evaluation of acute and chronic toxicity tests with larval striped bass. Aquatic Toxicology and Risk Assessment: Fourteenth Volume, ASTM STP 1124, M. A. Mayes and M. G. Barron, Eds., American Society for Testing and Materials, Philadelphia: 193-211.

For acute testing, the mortality of 6 to 45-day post hatch Striped bass (Morone saxatilis) was evaluated when exposed to varying concentrations of Bolero 8EC (85.2% thiobencarb) for 96 hours under flow through conditions. There were five concentrations for the test substance and two replicates for each test concentration and negative control. Solvents were not needed to prepare solutions with the formulation and there was no solvent control. Observations on mortality were made every 24 hours and the dead test subjects were counted and removed.

b Control mortality was between 11-20%,

Survival data were analyzed using a binomial chi-square test. Length and weight data were analyzed using the Kruskal-Wallis test and the Dunn multiple-comparison test was used to compare treatment means. Statistical significance for NOAEC determination for both striped bass and Chinook salmon was p < 0.05.

Chronic test results for striped bass exposed to thiobencarb are reported in Table 5 of the test report. The lowest early life-stage chronic toxicity NOAEC for survival was a mean value of 21 µg/L (LOAEC = 36 µg/L, measured). This test was performed by ATL in 1989 on 1-hour prehatch striped bass and can be used quantitatively with the note that it likely underestimates risk. In the test performed on 9-day post hatch bass, significant effects on survival were observed in all treatments and a NOAEC could not be determined (NOAEC/LOAEC = 21/ 36 µg/L). For the test beginning with 8-day post hatch bass the NOAEC and LOAEC were 58 and 91µg/L, respectively. Effects on dry weight were statistically different from controls at all treatment levels and a NOAEC could not be determined. All endpoints could underestimate risk because hatchability was not evaluated and a NOAEC could not be determined. However, the results can be used quantitatively as the available results are lower than values in registrant submitted studies. This test may not be used to fulfill test guidelines.

The Chronic NOAEC for Chinook salmon was 28 µg/L for growth (wet weight and standard length) and 140 µg/L for survival. The Chinook salmon NOAEC is not lower than the registrant submitted endpoint. These endpoints may be used quantitatively; however, it should be noted that they may underestimate risk because hatching success was not measured in the study. Therefore, these results may not be used to fulfill guideline requirements.

This acute, 96-hour freshwater fish toxicity study (Test No. 9) has a lower LC₅₀ than the registrant submitted study. The lower LC₅₀ value was 440 ug/l and may reveal increased risk in the analysis of the ecological risk assessment. The chronic striped bass study is not a complete early-life stage study, but is a scientifically sound methodology. The chronic endpoints may be useful quantitatively in a risk assessment with the note that risk may be underestimated due to the test not examining hatching success and the lack of a NOAEC for growth. The chronic Chinook salmon study is not lower than registrant submitted endpoints, but may be valuable in establishing a species-specific acute to chronic ratio.

The following additional limitations were observed in the study design and reporting of data.

• 
  No raw data were provided.
  
• 
  Environmental parameters during the study were not specifically described, such as pH, dissolved oxygen, and nominal versus measured thiobencarb concentration. The photoperiod was not described. It may be assumed that the photoperiod was consistent with those required by the ASTM method cited.
  



Addendum to OLRS for malathion (PC 057701)

b Control mortality was between 11-20%,

Adult female fish (80-90g body weight and 170-205mm length) were collected from the Bhubaneswar fish market. They were acclimated in the laboratory for two weeks and fed minced goat liver every other day. Technical malathion (purity not reported, Pest Chem. India) was used and stock solutions were prepared in acetone. The 96-hr LC50 for malathion was reported to be 448 ppm.

[Major] Given that 448 mg/L is above the water solubility of malathion, and there is insufficient information available in study regarding measured concentrations, the results were not used in the SSD. The focus on the study was using a sublethal concentration of malathion to examine gonadal and brain effects. The acute LC50 value was calculated in an effort to set the sublethal concentrations.

1. 
   Solvent concentrations were not reported
   
2. 
   Test concentrations were not reported to be measured in this study
   
3. 
   Unknown if control used and if there was any control mortality.
   
4. 
   Fish obtained from local fish market, so previous exposure to potential contaminants unknown. This is also true for food source (minced goat liver).
   

The acute toxicity of malathion to glochidia, juvenile, and adult freshwater mussels was determined at pH 7.5 in soft water and at pH 7.9 in moderately hard reconstituted fresh water at 25°C and 32°C. Nine species were tested in one or more life stage. Species tested included: Villosa lienosa, V. villosa, Utterbackia imbecillis, Megalonaias nervosa, Lampsilis teres, L. siloquoidea, L. straminea claibornensis, L. subangulata, and Elliptio icterina. Glochidia tests were conducted for 4, 24, or 48 h, while juvenile and adult exposures lasted 96 h. The purity of malathion was 96% and was dissolved in analytical grade acetone. Solvent controls were added to test design in addition to a dilution water control. Dilution water was prepared by adding appropriate salts to deionized water (following EPA methods) or by diluting well water. The quality of the dilution water was measured several times during its use. Water was filtered (0.2 um) and aerated prior to use. Tests were conducted under a 12:12 L:D photoperiod. Test solutions were measured and showed little dissipation 24 and 96 hours after preparation at 25°C. LC50 values were calculated using probit analysis and were based on initial malathion concentrations.

Mature glochidia (0.2-0.4 mm diameter) were collected from two or three adult female mussels and pooled and placed into well polystyrene dishes. Two sets of three or four replicates (each containing 50 to 100 glochidia) were used. Five test concentrations were tested (same for adult mussel testing). Mortality was observed at either 24 or 48 hours for all species except, for Lampsilis teres which were only observed for 4 hours. Control mortality of 20% was used as a maximum allowable threshold (10% allowable for other studies). Juvenile organisms used in testing were collected from infecting host fish in laboratory. Juveniles were randomly distributed to glass petri dishes, with 2-4 replicates per concentration and 10-20 juveniles, dependent on availability, and observed for 96 hours under static conditions. LC50 values calculated using probit analysis. For the adult mussel tests, two to four test concentrations were used with 5-10 mussels per concentration, and were observed for 96 hours.
Results:

For the glochidia tests, control mortality often exceeded 20% in tests conducted at 32°C and therefore LC50 values were not calculated for those studies. LC50 results for glochidia tested under different water pHs and temperatures are presented in Table 1 (from Table 2 in study report).

Results for juvenile mussels in moderately hard water and soft water are presented in Table 2 and 3, respectively (presented in Table 3 and 4 in study report).

For the adult mussel toxicity studies, 50% mortality was not observed for V. lienosa, U. imbecillis, or E. icterina up to 350 mg/L after 96 hours. However, sublethal effects were observed in these studies including slower or no shell closure and large amounts of mucus at concentrations of 150 mg/L and greater.

1. 
   Test material source was not reported, and unclear if impurity profile is reflective of current standards.
   
2. 
   Solvent concentrations were not reported (test solutions prepared by serial dilution)
   
3. 
   While examination of test solution recovery (concentration over time) were examined in previous work, concentrations were not measured at test termination in these studies. This is of potential greater uncertainty in studies conducted at 32°C since pre-work was at 25°C.
   

Panda S.; Sahu S.K. (1999). Effects of Malathion on the Growth and Reproduction of Drawida willsi (Oligochaeta) Under Laboratory Conditions. Soil Biol. Biochem. 31: 363-366.

This study conducted toxicity tests (mortality) and sublethal tests (growth and reproduction) on earthworms, Drawida willsi, to malathion.

Results of the 96-hr toxicity test are shown in Table 1.

In regards to the chronic study, a significant decrease in growth was reported after 15 days which continued until day 60, after which no significant difference was observed from 75 days until test termination (105 days). It was reported that increased growth in the controls was 84% versus an increase of 27 and 4% at 2.2 and 4.4 mg/kg, respectively, after 15 days of exposure. It the chronic study, control worms reached the immature and adult stage after 15 and 45 days, respectively, whereas, in the 2.2 and 4.4 mg/kg, the immature and adult life stage was not reached until day 30 and 60 or 45 and 75, respectively. Significant differences in the rate of reproduction were reported between the control and experimental set at 75 and 90 days, but not at 105 days. The rate of reproduction in the controls at 75 and 90 days was 2.1 and 2.0 cocoons/adult (average of 5 earthworms), and was 1.9 and 2.0 cocoons/adult in the 2.2 mg/kg and was 1.4 and 1.6 cocoons/adult in the 4.4 mg/kg.

• 
  Since the test material was sourced in India, it is uncertain if the formulation and impurity profile are reflective of current standards.
  
• 
  Variability in the treatment means were not reported (e.g., standard error, standard deviation or confidence limits);
  
• 
  The ANOVA tests indicated that there were significant differences between the treatment groups, but it in itself won’t tell which groups are different. Based on the description of the statistical analysis and reported results, it is unclear if additional statistical tests were performed (e.g., a pair-wise test). Based on the reported values, it is plausible that growth was significantly affected in both concentrations and reproduction in the high treatment, but this cannot be confirmed.
  

• 
  Raw data were not available to confirm calculations and statistics.
  
• 
  It is uncertain whether data was corrected for percent technical (in the absence of additional information, the reviewer corrected for % a.i.);
  
• 
  Control mortality in the acute LC₅₀ studies were not reported;
  
• 
  While the solvent was reported to allow to evaporate, a no-solvent control was not added to evaluate for possible solvent interferences in test results.
  

Sahu, S.K., Senapati, B.K., 1988. Alternative proposals for quantification of reproduction in a tropical earthworm. Tropical Ecology 29, 6-14.

Sahu, S.K., Mishra, S.K., Senapati, B.K., 1988. Population biology and reproductive strategy of Dichogaster bolaui (Oligochaeta: Octochaetidae) in two tropical agroecosystems. Proceedings Indian Academy of Sciences (Animal Science) 97, 239-250.

Senapati, B.K., Dash, M.C., 1979. Laboratory observation on the rate of cocoon production of Drawida willsi (Oligochaeta). Journal of Comparative Physiology and Ecology 4, 110-112.

Snedecor, G.W., Cochran, W.G., 1967. Statistical Methods. Iowa State University Press, Ames.
Primary Reviewer: Stephen Carey, Biologist, US EPA, Office of Pesticide Programs

Secondary Review: Amy Blankinship, Chemist, US EPA, Office of Pesticide Programs
Open Literature Review Summary
Chemical Name: Malathion

CAS No: 121-75-5

PC Code: 057701

ECOTOX Record Number and Citation:
E72010

Caron, D.M. (1979). Effects of Some ULV Mosquito Abatement Insecticides on Honey Bees. J. Econ. Entomol. 72: 148-151.

This paper from University of Maryland reports results of field toxicity tests of malathion, pyrethrum, and naled formulations to caged honeybees (Apis mellifera L.) and as bee colonies that were conducted at measured distances from the spray site receiving both ULV and diluted sprays.

Results showed that bee mortality decreased rapidly with increased distance. Percent mortality of caged bees at measured distances is shown in Table 1; while mortality of caged bees at varying distances up to 300 m in the 4 communities is shown in Table 2. It was noted by the study authors, that the results of the field (varying distance up to 300 m) study were similar to the results at 30 and 60 m in the controlled distance study.

In the bee community study where colonies were placed into communities receiving mosquito abatement sprays, the study authors noted that after the 5 applications of ULV pyrethrum, mortality of bees were averaging less than 100 bees/day (normal losses), however, mortality after malathion (57% EC) greatly increased (maximum mortality of approximately 1000 bees based on Figure 1 in paper). In the other colony study, malathion was applied both during the day and night. Mortality after the nighttime applications were low (both considered typical daily loss), and mortality after daytime applications were greater (maximum mortality of approximately 1500 bees based on Figure 2 in paper).