Description of Use in Document

Other limitations of the study included: 1) only mean values were reported without any understanding of the variability (e.g., 95% confidence intervals, standard deviations) around that value; 2) acetone was used to prepare the test concentrations, however, it is unknown if the control contained acetone or was a dilution water control only; 3) test concentrations were not measured; 4) given that fish died in the experiment (although unsure of how many fish and in what treatments), there is uncertainty in how this may have affected measurements as fish were likely stressed prior to death.

Drummond RA and Olson GF. 1974. Cough Response of Brook Trout (Salvelinus fontinalis) Exposed to Four Metals and Four Pesticides. Draft Manuscript from A. Jarvinen files, U.S.EPA, Duluth, MN : 10 p.

Yearling brook trout (Salvelinus fontinalis) were exposed to metallic and pesticide compounds and cough response was measured. The abstract reported that the lowest concentration of each compound to cause a significant increase in cough frequency was compared to available data regarding long-term effects.

Kundu, C.R. and S. Roychoudhury. 2009. Malathion-induced sublethal toxicity on the hematology of cricket frog (Fejervarya limnocharis). J. Environ. Sci. Health Part B. 44:673-680.

Cricket frogs, Fejervarya limnocharis, (6.5-6.6 g) were collected from a paddy field (India), acclimated for 10 days, and were exposed to 0.006 ppm malathion (50EC) for 240 hours and hematological parameters were evaluated. While specific testing details were not provided for the definitive test, details on the preliminary experiment were provided and the reviewer assumes these conditions applied to the definitive test as well. Test solutions were renewed every day and the test material was dissolved in acetone. A single test concentration (0.006 ppm malathion 50EC) and a control were used. Ten animals were placed into each replicate (glass jars with 5-L capacity) with three replicates per treatment. Temperature, pH and dissolved oxygen were measured. Blood samples were collected (from dorsal aorta) from 6 individuals at 24, 48, 72, 96 and 240 hours. Erythrocytes and leucocytes were counted (using Neubauer’s improved haemocytometer) and differential leucocyte count was also performed (using Wright’s stain). The length and breadth of erythrocytes were measured using ocular and stage micrometer. Haemoglobin and packed cell volume (mg/100mL) were also measured. Statistical analyses completed using Student’s t-test and Scheffe’s test.

Total erythrocyte counts were significantly lower and total leucocyte counts were significantly greater at all time points in the malathion treatment compared to control (Table 1 and 2 below, copied from paper). Additionally, neutrophil counts were significantly lower and eosinophil and lymphocyte counts were significantly greater at all time points for the malathion treatment compared to control. The length, width and surface area of erythrocytes were reduced compared to the control at 72 and 96 hours; surface area was still significantly reduced at 240 hours. Hemoglobin and packed cell volume/content (mg/100mL) were significantly lower at all time points. Other morphological changes in erythrocytes and leukocytes were also observed in the blood films (e.g., dissolution of nuclear material in erthyrocytes, leucocyte degeneration) of the malathion-treated animals.

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  acetone was used to prepare the test concentrations, however, it is unknown if the control contained acetone or was a dilution water control only. Solvent amount used in the test solutions were not reported, and since only one control was used, it is not known if the solvent may have influenced the measured endpoints;
  
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  test concentrations were not measured;
  
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  it was not definitively stated that the conditions reported in the preliminary test were the same in the definitive test;
  
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  animals collected from a paddy field, and therefore, prior exposure to potential contaminants is unknown;
  
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  life-stage of the animals were not reported;
  
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  the reviewer is uncertain if the reported test concentrations were adjusted for purity (ECOTOX assumes they were);
  
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  The formulation product name is not given, and there is uncertainty in whether the impurity profile is reflective of the current standards.
  

Patil, V.K., and M. David. 2010. Behavioral and morphological endpoints: as an early response to sublethal malathion intoxication in the freshwater fish, Labeo rohita. Drug Chem. Toxicol. 33(2): 160-165.

Carp, Labeo rohita, fingerlings collected from India State Fisheries department were acclimated in laboratory for 30 days in cement tanks and then acclimated for 20 days at 24±1˚C in glass aquaria. Fish (5 ± 1g, 7.5±0.25cm) were held in dechlorinated tap water with the following water characteristics: temperature, 24 ± 2°C; pH, 8 ± 0.2 at 24°C; dissolved oxygen, 9.6 ± 0.8 mg/L; carbon dioxide, 6.3 ± 0.4 mg/L; total hardness, 23.4 ± 3.4 mg as CaCO3/L; phosphate, 0.39 ± 0.002 μg/L; specific gravity, 1.001; and conductivity less than 10 μS/cm, and a 12–12-hour photoperiod. Fish were fed dry feed pellets (Nova; Aquatic Feed Pvt. Ltd) up to 2 days before initiation of the acute toxicity test. Toxicity tests were conducted with malathion (50% EC a.i. 50g/100mL; purchased from local market in India). In the acute toxicity test, solutions were renewed daily and mortality was monitored for 96 hours. Rangefinding was reported as using batches of 10 fish in 20L of solution. The test concentrations in the definitive acute test were reported to be between concentrations resulting in 0% and 100% mortality (actual test concentrations not reported). An additional test was conducted at 1/10^th the calculated acute LC50 value (0.45 µg/L) where fish were exposed for 1, 5 or 15 days and then placed in dilution water for 15 days. Fish were fed and were observed for behavioral responses and morphological deformities. Brain and muscle acetylcholinesterase (AChE) were measured (using method by Ellman et al. 1961; spectrophotometer). A control was reported to have been used in both tests. All reported concentrations are as nominal and it was not clear if concentrations and results were adjusted to account for percent malathion (50%).

The reported 96-hr LC₅₀ value was 4.5 µg/L (2.25 µg/L when adjusted for 50% malathion; mortality data not provided). No mortality was observed in the sublethal test. The control fish in the sublethal test were reported to have been behaving normally (actively feeding, alert to disturbances) and did not vary throughout the exposure period. In the malathion treatment (0.45 µg/L; 0.225 µg/L when adjusted for purity), fish were reported to display shoaling behavior (by the first day of exposure), erratic swimming, staying near bottom of tank, spread out (2x the area occupied in tank as compared to control), and loss of coordination. The study reported that fish became lethargic, restless, and secreted excess mucus over their body. A behavior reported as being easy to catch was reported on the first day of exposure and reported to continue during the non-exposure period. Caudal bending was also observed in the exposure and non-exposure periods. Feeding preferences and food consumption were also reported to be affected even in the non-exposure period. AChE activity was reported to be reduced during the exposure period (Figure 1 in paper).

Several papers similar to this study have been published by the authors. In looking at these additional papers, there is uncertainty in the reported test concentrations in this paper. The additional information in the other publications that lend support to this uncertainty are:

Three of these studies report the test concentrations in terms of µL/L. Density is not reported in any of these three studies. However, a density is reported in this paper and is 50 g/100mL (500 g/L). While the paper does not specify whether the density is for the active ingredient (malathion) or the formulation, it is common to report the density in terms of the a.i. on a commercial label. While the test concentrations in the fourth study are the same as this study, the test concentrations in that paper were only reported in the abstract and were only referenced as a lethal and sublethal concentration in the actual paper.

In regards to the esterase activities as different age groups (results reported for 28, 42, and 84 day old rats in addition to the 1, 6, 12, and 17 described above; details for these additional ages not reported; reviewer believes these discussions are in reference to a control or untreated group of rats). The study author reported that liver enzymes hydrolyzing BSChI and ASChI increased in activity during the first 7 days after birth and then remained constant IPA hydrolysis was greater than BSChI or ASChI and increased 28 days after birth. Protein concentrations generally remained constant over 84 day period. The study also reported that brain esterase activity for BSChI increased after 7 days after birth and then remained relatively constant. Brain activity towards ASChI also increased during first 7 days then increased further with age, and IPA hydrolysis rate increased during first 7 days but then declined from Day 13 on. There was a decrease in brain protein concentration during the first week after birth. For kidney, esterase activity towards BSChI and ASChI increased during the first 7 days and then decreased by Day 28 and remained constant. IPA gradually increased during first 3 weeks, declined and stabilized. Protein concentration in kidney gradually increased and then remained constant from day 13 to 84.