Chlorpyrifos

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish life cycle toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 204 (Fish, Prolonged Toxicity Test: 14-day Study)

Deviations:

yes

GLP compliance:

yes

Specific details on test material used for the study:

Substance ID: Dursban F
Batch#: Ek 880303344
Purity: 97.2% of chlorpyrifos

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

Tertiary butanol

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Test type:

flow-through

Water media type:

other: Ground water

Total exposure duration:

21 d

Hardness:

21. mg/L as CaCO3

Test temperature:

13.7 - 16.1°C

pH:

7.5 - 8.1

Dissolved oxygen:

>6.3 mg/L

Nominal and measured concentrations:

Nominal: 0.54, 0.97, 1.75, 3.1, 5.4 µg/L

Key result

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

> 3.2 - < 5.6 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

4 d

Dose descriptor:

LC50

Effect conc.:

> 5.6 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 1.8 - < 5.6 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

3.2 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.8 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

3.2 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Food uptake

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: swimming behaviour including loss of equilibrium

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.8 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Colour

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.93 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Mortality and growth rate

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1.6 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Mortality and growth rate

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.51 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Mortality, growth, behaviour, color, feeding and loss of equilibrium

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.93 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Mortality, growth, behaviour, color, feeding and loss of equilibrium

Validity criteria fulfilled:

yes

Conclusions:

21-day NOEC (RainbowTrout): 1.8 µg/L (Based on growth rate)
21-day NOEC (RainbowTrout): 3.2 µg/L (Based on mortality)
21-day NOEC (RainbowTrout): 1 µg/L (Based on food uptake)

Executive summary:

The study was conducted according to the guideline OECD 204 to evaluate the influence of test substance on growth rate and mortality of the fresh-water fish species when exposed for 21 days. The test was carried out with a flow-through system.

The concentrations tested were 0.56, 1.0, 1.8, 3.2 and 5.6 µg/L. No effect on mortality of test species was found at 3.2 µg/L and the lower concentrations tested. At 5.6 µg/L, all animals died during the last three exposure days. The 21 d LC50 was therefore estimated from the data to be greater than 3.2 µg/L and less than 5.6 µg/L, the 4d LC50 was > 5.6 µg/L. No significant effect on the instantaneous growth rate was found at 1.8 µg/L and the lower concentrations tested.

A significant effect on the instantaneous growth rate was found at 3.2 and 5.6 µg/L, at the latter concentration the weight of the animals decreased. These animals were weighed just after they died. The 21 d EC50 (E = growth rate) was estimated from the data to be greater than 1.8 µg/L and less than 5.6 µg/L.

The NOEC (no observed effect concentration) for the criteria investigated quantitatively (mortality and growth) was 1.8 µg/L and the LOEC (lowest observed effect concentration) 3.2 µg/L. 

In addition to the quantitatively determined effects on mortality and growth, effects on swimming, behavior, color, respiration and feeding were visually estimated. At 1.0 µg/L and the lower concentration tested no effects on these criteria were found at the end of the test. At 1.8 µg/L and the higher concentrations tested the fish were slow, dark, took less or no food and showed loss of equilibrium. These effects increased with increasing concentration and time. During the final days of the test white spots were seen on these fishes. Including these effects the NOEC was 1.0 µg/L and the LOEC 1.8 µg/L.

Assuming the active substance concentration during the test of 53% of the nominal value, the NOEC for mortality and growth would be 0.93 µg/L and the LOEC was 1.6 µg/L; the NOEC for mortality, growth, behavior, color, feeding and loss of equilibrium would then be 0.51 µg/L and the LOEC 0.93 µg/L.

Reference 2

Endpoint:

fish life cycle toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 72-5 (Fish Life Cycle Toxicity)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Sample ID: Chlorpyrifos (AGR 284109)
Lot#: Not reported
Purity: 99.7%

Analytical monitoring:

yes

Details on sampling:

Mean analyzed test substance concentrations (grand means of daily averages) determined for the individual nominal treatment levels of 63, 125, 250, 500, and 1000 ng/L were 82.6, 143.8, 300.1, 568.3, and 1093.2 ng/L dilution water, respectively. The overall mean accountability, [i.e. recovery], averaged across all treatment levels was 117.8 ± 8.3%.

Vehicle:

yes

Remarks:

Dillution water

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A proportional diluter system was used to achieve exposure concentrations. This system was designed to deliver five chemical concentrations (with a dilution factor of 0.5), and a water and solvent control. The solvent (acetone) was limited to no more than 0.1 mL/L. The system functioned as follows: a precision dosing system delivered the test chemical from a stock bottle to a mixing chamber, where it was actively mixed with dilution water using a recirculating pump. After mixing, the chemical solution was distributed to chemical cells; and, when the dilutor cycled, the solution from each chemical cell mixed with water from a respective water cell and flowed into splitting chambers. Splitting chambers have delivery tubes which run to each of the replicate test vessels at each concentration or control. Flow rate from these chambers was calibrated to approximately 2 L ± 200 mL per replicate per diluter cycle.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Source: Breeding unit, The Environmental Toxicology & Chemistry Research Laboratory Health and Environmental Sciences, The Dow chemical company, Midland, Michigan.
- Age at study initiation: Less than 24 hours old
- Method of breeding: Embryos were selected from stainless steel laying substrates placed in the breeding aquaria no more than 24 hours prior to the beginning of the test.
- Feeding during test : Yes
- Food type: Larvae were fed live, newly hatched, brine shrimp
- Frequency: Fed three times a day on normal work days and once or twice a day on weekends.

Test type:

semi-static

Water media type:

other: Laboratory water

Total exposure duration:

32 d

Post exposure observation period:

Till Day 216

Hardness:

73.8 mg/L CaCO3

Test temperature:

25.2°C

pH:

7.0 - 8.1

Dissolved oxygen:

7.8 mg/L (>92% saturation)

Conductivity:

181.5 mg/L CaCO3

Nominal and measured concentrations:

Nominal: 63, 125, 250, 500, and 1000 ng/L
Measured: 82.6, 143.8, 300.1, 568.3, and 1093.2 ng/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Test aquaria were constructed of glass and clear silicone adhesive and measure 46x46x22 cm deep. Each was provided with two 22x13x22 cm deep larval growth sections. The grow-out portion of each aquarium (33x46x22 cm deep) was designed to be partitioned into four spawning chambers, each measuring 16x13x22 cm deep. The partitions between all sections were provided with screens which allowed a free flow of water throughout the aquarium. The grow-out sections were provided with screen covered drains which maintained a water depth of approximately 16.5 cm.
- Aeration:
- Type of flow-through: Proportional diluter
- Renewal rate of test solution: Flow rate from these chambers was calibrated to approximately 2 L ± 200 mL per replicate per diluter cycle. Flow rate through each replicate provided at least five volume changes per 24 hours.
- No. of organisms per vessel: The embryos were divided equally between 2 embryo cups, each containing 20 embryos, set in each of the two replicate aquaria at each concentration.
- No. of vessels per concentration: Two replicates
- No. of vessels per control: Two replicates
- No. of vessels per vehicle control: Two replicates

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water used in the laboratory was pumped from the upper Saginaw Bay of Lake Huron. The water was limed and flocculated with ferric chloride by the City of Midland Water Treatment Plant. Prior to use the water was sand filtered, carbon filtered, UV irradiated and the pH adjusted with C02. The water was monitored weekly for hardness, alkalinity, pH, and conductivity, quarterly for selected inorganics, total suspended solids, total dissolved solids, and total organic carbon (TOC), and twice annually for selected organic compounds, pesticides and polychlorinated biphenyls' (PCBs).
- Total organic carbon: Not detected
- Metals and inorganics: The metals available in standard dilution water were aluminum (32 µg/L), ammonia (32 µg/L), boron (36 µg/L), bromide (22 µg/L), calcium (16000 µg/L), fluoride (150 µg/L), iron (23 µg/L), magnesium (7800 µg/L), phosphorous (450 µg/L), potassium (1100 µg/L), silicon (700 µg/L), sodium (5090 µg/L), sulfur (6200 µg/L), strontium (85 µg/L)
- Pesticides: Aldrin, Aplha-BHC, Gamma-BHC, chlordane, heptachlor, heptachlor epoxide, hexachlorobenzene were was detected at concentration of <0.05 µg/L, 4, 4-DDD, 4,4-DDE, dieldrin, endrin, PCB-1242, PCB-1248, PCB-1254, PCB-1260, diazinon, disulfoton, malathion, parathion ethyl, parathion methyl were detected at concentration <0.1 µg/L, methoxychlor was detected at concentration of <0.5 µg/L, toxaphene was detected at concentration of <2.0 µg/L
- Chlorine: present as 17300 µg/L of chloride
- Alkalinity: 49.1 mg/L CaCO3
- Conductivity: 181.5 µmhos/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: Weekly

OTHER TEST CONDITIONS
- Adjustment of pH: Yes with CO2
- Photoperiod: 16-h light and 8-h dark photoperiod was maintained throughout the test with a 15 minute transition between dark and light.
- Light intensity: 1025 - 1252 Lux

EFFECT PARAMETERS MEASURED :
Initial early life-stage exposure (Parental generation): Embryos were observed daily; dead embryos and larvae were counted and removed at each observation. Upon completion of hatching, the total number of larval in each replicate, including those dead or deformed, were counted. Dead or deformed larvae were subtracted from the total to determine the number of normal larvae at hatch. Also the percent of embryos that hatched and the day to mean hatch at each replicate was calculated. After hatching, each replicate group of larvae including deformed individuals were released into the larval incubation chambers. Weekly mortality and sublethal effects observations as well as water quality observations were recorded during this phase of the study.
Larval-Juvenile exposure: Mortality and sublethal effects and water quality were recorded weekly during the larval-juvenile exposure period.
Juvenile-Adult Exposure: Mortality and sublethal effects and water quality were recorded weekly during the juvenile-adult exposure period.
Reproductive effects observations: Secondary sex characteristics were expressed by many fish by day 90 and embryos were detected on some spawning substrates on day 104. From day 105 through at least day 200 the following reproductive parameters were followed, total spawns, eggs laid, hatchability of embryos and day-to-final hatch of selected embryos. These data were converted to mean spawns per breeding unit, mean number of eggs laid per treatment level, mean number of eggs per spawn per treatment level and mean percent hatch. In several instances embryos set for hatchability evaluation washed out of the embryo cups or were lost upon transfer. The number of eggs actually observed were used in the analyses of hatching success.

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

568 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Observed effects at each concentration for each observation time: General observations: Sublethal effects noted were curved spine of one fish at the 1093 ng/L treatment level, and a fish with a curved spine, one with reduced growth of an operculum and another with a small abdominal growth, all in the water controls. At termination of the non-breeding fish one fish at the 568 ng/L treatment level was observed with a stumpy tail. During hatching-success observations, made during the reproductive phase. the primary effects noted were lack of fertilization and small under-developed larvae which may indicate premature hatch. These effects were observed across treatment levels and controls.
Parental Early life-stage exposure: There was no statistical difference in the percent hatch or in the percent normal at hatch. The day-to-mean hatch was day four at all treatment levels. However, the percent hatch on day four was significantly decreased at the 1093 ng/L and 568 ng/L treatment levels when compared to the acetone control (not the water control). These differences were not evident on day five.
Larval-Juvenile Exposure: There was no statistically significant difference in growth of the larvae after 31 days of exposure. A statistically significant decrease in growth, when compared to the acetone controls but not the water controls, was noted at the 300 ng/L level on Day 61. However, this effect was not observed at test substance treatment levels above or below this concentration. Analysis of the Day 61 data also indicated that the water control fish were significantly smaller than the acetone control.
Juvenile-Adult Exposure: The breeders were sacrificed on Day 216 and the growth of these fish assessed. There was no statistically significant difference in either length or weight of these fish when compared to the acetone controls.
- Concentrations that produce lethal or other effects: Parental Early life-stage exposure: On days 12, 19, and 25 there was a significant increase in mortality at the 1093 ng/L treatment level when compared to both the water and acetone controls. No other significant increase in mortality was detected.
- Cumulative mortality at each concentration and for each recommended observation time if possible: Given under "Any other information on results incl.table" under summary of parental generation mortality.
- Reproductive observations:
The reproductive data were variable. The mean number (± standard deviation) of spawns per breeding unit ranged from 5.4 (±5.8 ) for the acetone control to 11.4 (±7.2) for the water controls. This parameter varied non-systematically among the treatment groups, ranging from 6.6 (±4.9) at 568 ng/L to 9.1 (±6.3) at the 144 ng/L treatment level. Similar variation was noted for the mean number of eggs per spawn per breeding unit. These data range from 68.8 (±52.5) at 83 ng/L to 113.5 (±46.0) for the water controls. Average egg production was also variable, with a low of 2312 (±497) at 568 ng/L to 5041 (±2050) for the water control. Examination of these data indicates that the water controls had relatively higher reproductive performance, however, the reproductive data for the acetone controls was consistent with the remainder of the treatment levels and there was no statistically significant treatment-related effects on any of the reproductive parameters analyzed.
F1 Generation Early life-stage Exposure: Because of the lack of synchrony in egg production, the F1 generation embryo-larval study was initiated on day 200 for the 83 and 568 ng/L treatment levels, day 202 for the water control and the 144 ng/L treatment levels, day 205 for the 1093 ng/L treatment level, day 209 for the 300 ng/L treatment level, and day 210 for the acetone control. The day-to-mean hatch was day 3 for the 1093 ng/L treatment level and the water control, and, day 4 for the remaining treatment levels and the acetone control. No statistical difference was demonstrated between the treated groups and the controls for the percent hatched, and percent normal at hatch. Analysis of variance calculations demonstrated that the water and acetone controls were statistically different for survival on days 14, 19, 22, 27 and 32. Therefore, mortality data for the treated groups were compared with each control separately. Mortality in the 1093 ng/L treatment group was statistically higher on days 8, 11, 14, 19, 22, 27, and 32. At the termination of the exposure period there was no significant difference in larval growth (as indexed by length and weight) between any of the treatment groups and the controls.

Reported statistics and error estimates:

The first step in the analysis was to make scatter plots of the responses versus the concentration, then to compare the water and vehicle (acetone) controls. If the controls were not statistically significantly different from each other, they were combined for the subsequent analysis of all test material concentration levels. The data for all endpoints, except the variables (mortality and terata) in the form of a proportion, were first examined for normality, using the Shapiro-Wilk's test, at a type I error rate of 0.01. If the data were not normally distributed, the logarithmic, inverse, and square root transformations were tested sequentially, to search for a normalizing transformation. Next, the raw data or the transformed variables were tested for homogeneity of variance using Bartlett's test. The Bartlett's test was conducted at a type I error rate of 0.01. If the raw data, or a transformation, could be considered normally distributed and homogeneous, it was analyzed using Analysis of Variance (ANOVA) techniques, followed by a one-sided Dunnett's test (at an alpha level of 0.05) to compare each concentration level with the control. Data in the form of proportions was transformed by the arcsine square root followed by ANOVA and Dunnett's test. For several of the endpoints, it was not possible to find a normalizing transformation, and the small number of replicates were inadequate for a nonparametric analysis, these variables were analyzed parametrically. The software used for these analyses was the SAS® statistical package running on an IBM mainframe computer.

Table1: Summary of parental generation mortality

Concentration (ng/L)	Replicate	Day 31	Day 61	Day 97	Day 111	Day 145	Day 180	Day 200	Day 208	Day 216
1093	A	0	0	0	0	0	0	0	0	0
	B	2	2	2	4	5	6	6	6	6
568	A	0	0	0	0	0	0	0	0	0
	B	0	0	0	0	1	1	1	1	1
300	A	2	2	2	2	2	2	2	2	2
	B	0	0	0	0	0	0	0	0	0
144	A	0	0	0	0	0	0	0	0	0
	B	0	0	0	0	0	1	1	1	1
83	A	1	1	1	1	2	2	2	2	2
	B	0	0	0	0	0	0	0	0	0
Water control	A	1	1	1	1	1	1	1	1	1
	B	0	0	0	0	0	1	2	3	3
Acetone control	A	1	2	2	2	3	4	4	4	4
	B	0	0	0	0	0	1	1	1	1

Conclusions:

32-Day NOEC (Fathead minnow): 568 ng/L (Based on mortality)

Executive summary:

The study was conducted according to EPA guideline 72-5 to evaluate the hazard from prolonged pesticide exposure on fish reproduction and other life stages. This test was initiated with < 24 h old embryos and carried through 32 days of the F1 generation. Embryos and fish were exposed to 83, 144, 300, 568 and 1093 ng/L test substance, a carrier, and a water control. End- points evaluated were embryo hatchability and larval survival and growth of the parental generation, reproductive performance of parental generation, which included analysis of mean spawns per breeding unit, mean number of eggs laid per treatment level, mean number of eggs per spawn per treatment level and embryo viability; and, larval growth and survival of the F1 generation. Evaluation of the results of this study indicate that mortality was the most consistently sensitive toxicity end-point. Mortality was increased at the 1093 ng/L treatment level in both the parental and F1 embryo-larval exposures. The no observed effect concentration (NOEC) was 568 ng/L. Larvae less than 25 days of age were the most susceptible life stage. Statistically significant effects on growth were transient and nonsystematic in nature and and statistically significant effects on reproductive end-points were not observed.

Reference 3

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 229

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Substance ID: Chlorpyrifos Technical
Lot Number KC28161419 (TSN101285)
Purity 98.8%

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

Laboratory dilution water

Test organisms (species):

Pimephales promelas

Test type:

flow-through

Water media type:

other: Laboratory dilution water

Total exposure duration:

21 d

Hardness:

Pre-exposure phase: 66 - 70 mg CaCO3/L
Exposure phase: 60 - 72 mg CaCO3/L

Test temperature:

Pre-exposure phase: 24 - 25°C
Exposure phase: 25 - 26°C

pH:

Pre-exposure phase: 168 - 181 µS/cm
Exposure phase: 164 - 209 µS/cm

Dissolved oxygen:

Pre-exposure phase: 7.0 - 8.1 mg/L
Exposure phase: 7.0 - 7.7 mg/L

Conductivity:

Pre-exposure phase: 168 - 181 µS/cm
Exposure phase: 164 - 209 µS/cm

Nominal and measured concentrations:

Nominal: 0 (water control), 1.2 (24 hr), 1.2 (48 hr), 1.2 (96 hr), and 1.2 (192 hr) μg/L
Measured:

Key result

Duration:

35 d

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks on result:

other: There were no significant treatment-related mortalities in this study, suggesting that the concentrations (1.10 – 1.22 μg/L) and exposure durations (24 – 192 hours) of test substance used in the present study were not overtly toxic to fathead minnows.

Key result

Duration:

35 d

Conc. based on:

test mat.

Remarks on result:

other: There were no significant differences between control and test substance-exposed fish in regard to fertility or fecundity measurement indicating 1.10 to 1.22 µg/L at 21 to 192 h of test substance did not affect reproduction

Validity criteria fulfilled:

yes

Conclusions:

- There were no indications of abnormal fish behaviors or appearances, and there were no significant treatment-related mortalities in this study, suggesting that the concentrations (1.10 – 1.22 μg/L) and exposure durations (24 – 192 hours) of test substance used in the present study were not overtly toxic to fathead minnows.
- There were no significant differences between control and test substance-exposed fish in regard to fertility or fecundity measurements, indicating that the concentrations (1.10 – 1.22 μg/L) and exposure durations (24 – 192 hours) of test substance used in the present study did not affect fathead minnow reproduction

Executive summary:

The study was conducted according to guideline OECD 229 to evaluate modified fish short-term reproduction assay using the fathead minnow when exposed for 21-days. During pre-exposure phase, the test species was given only water for 14 days. The test type used was flow-through in both pre-exposure and exposure phase. The nominal test concentrations were 0 (water control), 1.2 (24 hr), 1.2 (48 hr), 1.2 (96 hr), and 1.2 (192 hr) μg/L. The mean measured concentrations of test substance were <LLQ (water control), 1.10 (24 hr), 1.22 (48 hr), 1.22 (96 hr), and 1.15 (192 hr) μg/L.

Based on mean measured concentrations, the results showed that there were no significant differences in fish survival between control and test substance treatment groups. Throughout the entire exposure period, there were a total of six fish mortalities (three in the controls, one in the 24 hr treatment, one in the 48 hr treatment, and one in the 192 hr treatment). Additionally, there were no indications of treatment-related abnormal behaviors or appearance, indicating that concentrations of test substance used in the present study were not overtly toxic to fathead minnows. At the end of the study, there were no statistically-significant differences (α = 0.05) between control and test substance-exposed fish in regard to fertility or fecundity measurements. Additionally, there were no statistically significant differences (α = 0.05) between controls and test substance treatment groups in regard to male and female fish lengths at the end of the 21-day study. Male wet weights in the 96 hr treatment group were significantly reduced compared to controls; however, there was no significant difference between controls and the 192 hr treatment group in regards to male wet weight. Female fish wet weights in the 192 hr treatment were significantly reduced compared to controls. The wet weights of female fish in the 192 hr treatment group were, on average, reduced 10% compared to control female wet weights.

Reference 4

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Remarks:

Short-term Reproduction assay

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 229 (2009)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OPPTS 890.1350 (2009)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Substance ID: Chlorpyrifos
Lot#: KC28161419 (TSN101285)
Purity: 99.8%

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

Laboratory dilution water (Lake huron water)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
continuous-flow diluter system was used to provide each concentration of the test substance and a negative (dilution water) control to the replicate vessels. The diluter system was operated as follows, for each treatment level, a peristaltic pump (e.g. Rainin Instrument, LLC, Oakland, California) continuously delivered a concentrated test substance stock solution at a designated rate from a stock vessel to a stainless steel mixing chamber where it was mixed with dilution water; no test substance was added for the water control test level. The dilution water flow and resulting test solution volume in the mixing chamber was maintained using a stainless steel float valve. The test solution was gravity fed from the mixing chamber and split equally to replicate test vessels (contained in a temperature controlled water trough) via a Teflon manifold with Teflon delivery tubing at a flow rate of 90 ± 9 ml/minute. This flow rate provided approximately 13 volume turnovers per day for each replicate test vessel. The target flow rate of 90 ml/minute was greater than the USEPA recommended flow rate of 45 ml/minute and was chosen in an effort to help maintain test concentrations during the exposure.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnows
- Source: New England Bioassay, Inc., Manchester, Connecticut, U.S.A.
- Age at study initiation: Approximately 6 months old at the initiation of this study
- Length at study initiation: Male: 52.6 ± 2.04 - 55.1 ± 2.85 mm; Female: 43.0± 0.30 - 43.7 ± 0.96 mm
- Weight at study initiation: The average weight of male fathead minnows (N=60) that were used to stock aquaria for the pre-exposure phase of the study was 3.5 ± 0.4g (coefficient of variation = 10.0%), while the average weight of the female fathead minnows used to stock aquaria (N=120) was 1.7 ± 0.2 g (coefficient of variation = 10.0%).
- Method of breeding: During pre-exposure period, two male and four female fathead minnows were impartially assigned to each of the 30 test vessels, which were equipped with three breeding substrates.
- Feeding during test : During pre-exposure and exposure periods, fish were fed thawed frozen brine shrimp (Brine Shrimp Direct, Ogden, Utah) at least twice per day at a rate sufficient to promote active reproduction and maintain body condition.
- Frequency: Fish were fed atleast twice per day

ACCLIMATION
- Acclimation period: 23 days
- Acclimation conditions: 25 ± 2°C
- Type and amount of food: Fish were fed thawed frozen brine shrimp (Brine Shrimp Direct, Ogden, Utah).
- Feeding frequency during acclimation: Atleast twice per day
- Health during acclimation: 2.3% mortality rate was observed

Test type:

flow-through

Water media type:

other: Laboratory dilution water (Lake huron water)

Total exposure duration:

36 d

Remarks on exposure duration:

36 days (15 days pre-exposure, 21 days exposure)

Hardness:

Pre-exposure phase: 60-70 mg CaCO3/L
Exposure phase: 0-74 mg CaCO3/L as measured weekly in the control and 3.02 μg/L treatments

Test temperature:

Pre-exposure phase: 24.3 - 25.3°C
Exposure phase: 24.8 - 25.4°C

pH:

Pre-exposure phase: 7.4 - 7.7
Exposure phase: 7.3 - 7.8

Dissolved oxygen:

Pre-exposure phase: 7.1 - 8.3 mg/L
Exposure phase: 7.0 - 9.1 mg/L

Conductivity:

Pre-exposure phase: 197 - 221 μmhos/cm
Exposure phase: 175 - 211 μmhos/cm as measured weekly in the control and 3.02 μg/L treatments

Nominal and measured concentrations:

Nominal: 0 (control), 0.2, 0.64, and 2 μg a.s./L
Measured:

Details on test conditions:

TEST SYSTEM
- Test vessel: Aquarium constructed of glass
- Size of vessel: 39 x 20 x 25 (L x W x H) cm
- Type: closed
- Material, size, headspace, fill volume: Each test vessel was an aquarium constructed of glass, sealed together with clear silicone adhesive, water depth: 13 cm, water volume: 10 L
- Type of flow-through: Peristaltic diluter
- Renewal rate of test solution: Flow rate: 90 ± 9 ml/minute. This flow rate provided approximately 13 volume turnovers per day for each replicate test vessel
- No. of organisms per vessel: Pre-exposure and exposure: 2 male and 4 females per vessel,
- No. of vessels per concentration (replicates): Pre-exposure: 30 replicate test vessels, exposure: Four replicate test vessels
- No. of vessels per control (replicates): Pre-exposure: 30 replicate test vessels, exposure: Four replicate test vessels
- Biomass loading rate: 0.1 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Upper Saginaw Bay of Lake Huron off Whitestone Point
- Particulate matter: Water was limed and flocculated with ferric chloride. Before use in the laboratory, the water was sand-filtered, pH-adjusted with gaseous CO2, carbon-filtered, and UV-irradiated
- Culture medium different from test medium: No
- Temperature: 25 ± 1ºC
- Dissolved oxygen: >60% air saturation (4.9 mg/L)
- pH: 6.5 - 9.0
- Intervals of water quality measurement: The water is typically monitored weekly for pH, alkalinity, hardness and conductivity. Periodically, the water is analyzed for total organic carbon (TOC), total suspended solids (TSS), pesticides, organics, metals and other organics.

OTHER TEST CONDITIONS
- Adjustment of pH: pH adjusted with gaseous CO2
- Photoperiod: 16 hr light:-8 hr dark
- Light intensity: 540 – 1080 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Pre-exposure period: Following the pre-exposure period, fecundity was calculated for each spawning group based upon the egg counts for the previous seven days. Only test vessels that exhibited acceptable spawning activity (i.e. spawning events at least wo times in the immediately preceding 7 days and egg production exceeding 15 eggs/female/day/replicate vessel), had no incidences of mortality or adverse effects and displayed the correct sex ratio (2 males and 4 females) were carried forward into the exposure period of the study.
Exposure period: Fish observations were made daily during the test, and any mortality or external abnormalities (e.g. hemorrhage, discoloration) were noted. Any abnormal behavior relative to the controls (e.g. hyperventilation, loss of equilibrium, uncoordinated swimming, atypical quiescence and feeding abstinence) was also noted when observed. Dead fish were removed when observed and were not replaced.
Daily inspections of the spawning substrate (including spawning tile and screened tray) were performed in each replicate test vessel. For each test vessel, the number of eggs laid (defined as the total of the eggs adhering to the spawning tile and those on the screened tray) and the number of these eggs that were found to be infertile were enumerated and recorded on a daily basis. Clean substrates were added to the test vessels to replace any that were removed for egg counts. Eggs were deemed infertile if they appeared opaque or clear with a white dot where the yolk had precipitated.
Termination of exposure: On day 21 of the exposure, fish were observed in situ for behavioral and secondary sex characteristics prior to their removal from the test vessel. Characteristics of particular importance included the presence or absence of coloration patterns (i.e. presence or absence of vertical bands, dark or light), dorsal fat pad, and ovipositor.

Reference substance (positive control):

no

Key result

Duration:

36 d

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Remarks on result:

other: No statistically significant differences in mortality among control and test substance exposed fish upto highest concentration tested (3.02 µg/L).

Key result

Duration:

36 d

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Remarks on result:

other: No statistically significant changes in wet weight or length among control and test substance-exposed fish up to the highest concentration tested (3.02 µg/L).

Details on results:

- Observed effects at each concentration for each observation time:
Fecundity and Fertility:
Pre-exposure: At the end of the two week pre-exposure period (15 days), fecundity measurements for the previous seven days across all replicate vessels (N=24) ranged from 9.4 - 37.2 eggs/female/reproductive day with an average fecundity (± standard deviation) of 25.0 ± 6.5 eggs/female/reproductive day. Fecundity measurements for the previous seven days across all pre-exposure phase replicate vessels carried over for use in the exposure phase of the study (N=16) ranged from 20.3 - 37.2 eggs/female/reproductive day with an average fecundity (± standard deviation) of 28.1 ± 5.1 eggs/female/reproductive day.
Exposure period: During the exposure phase of the study, fecundity ranged from 2.4 to 37.3 eggs/female/reproductive day across all test groups (N=16), while fertility ranged from 95.5 to 100.0% (N=16). For the control groups, fecundity ranged from 30.0 to 37.3 eggs/female/reproductive day, while fertility ranged from 98.1 to 99.2%. All replicate vessels met the specified test validity (performance) criteria for the control group, i.e. spawning at least every 4 days or approximately 15 eggs/female/reproductive day and fertility greater than 95% (USEPA, 2009). There were no significant differences between control and treatment groups in the percentage of fertile eggs (P= 0.3661).
Weight and Length:
Pre-exposure: The average weight of male fathead minnows (N=60) that were used to stock aquaria for the pre-exposure phase of the study was 3.5 ± 0.4g (coefficient of variation = 10.0%), while the average weight of the female fathead minnows used to stock aquaria (N=120) was 1.7 ± 0.2 g (coefficient of variation = 10.0%).
Exposure period: Neither weight nor length measurements among male or female fish exhibited a monotonic dose response with increasing concentrations of test substance; therefore, length and weight data for both sexes were analyzed by the one-way ANOVA. According to the ANOVA, there were no statistically significant differences in length or weight among control and test substance exposed male fathead minnows (p= 0.5567 and p = 0.3097, respectively) and female fathead minnows (p = 0.7196 and p = 0.5418, respectively).
Gonado-Somatic Index (GSI):
The median GSI values for both male and female fish did not exhibit a monotonic dose response with increasing concentration of test substance; therefore, this parameter was tested for significance using the one-way ANOVA. No significant differences between control and test substance exposed treatments were found in male or female GSI values (p = 0.4137 and p = 0.9807, respectively).
Secondary Sex Characteristics:
At test termination, observations performed for presence and/or absence of dark coloration, vertical banding, fatpad and ovipositor in fish indicated no treatment related findings. There were no tubercles noted among female fathead minnows at the end of the exposure period. Male tubercle scores were evaluated with the multi quantal Jonckheere-Terpstra test. Tubercle score was not significantly different between control and test substance-exposed fish (p = 0.0674).
Vitellogenin (VTG):
The median male VTG as measured by ELISA (technical replicates) did not exhibit a monotonic dose response with increasing test substance concentrations. Due to non-normality of the data, a log transformation was performed prior to analysis using the one-way ANOVA. According to the ANOVA there was no significant difference (p = 0.7578) between control and test substance-exposed fish.
Median female VTG did not exhibit a monotonic dose response with increasing test substance concentrations. Therefore, the female data were also analyzed using the one-way ANOVA. According to the ANOVA there was no significant differences (p = 0.5361) between control and test substance-exposed fish.
Sex steroids:
The median male and female testosterone data did not exhibit a monotonic dose response with increasing test substance concentrations. According to the ANOVA, there was no significant difference between control and test substance-exposed fish for male and female testosterone levels (p = 0.2767 and p = 0.7863, respectively). The median male and female 17β-estradiol data did not exhibit a monotonic dose response with increasing test substance concentration. There was no significant difference between control and test substance-exposed fish for female 17β-estradiol levels (p = 0.7552). According to non-parametric Mann-Whitney Wilcoxon U test using a Bonferroni-Holm adjustment, there was no significant difference between control and test substance-exposed fish for male 17β-estradiol (p = 1.000).
Cholinesterase activity:
On a percentage basis, brain cholinesterase activity in female fish was reduced to 60%, 27% and 8% of the female control value by exposure for 21 days to 0.251, 0.812, and 3.02 μg/L test substance, respectively. Similarly, brain cholinesterase activity in male fish was reduced to 65%, 28% and 9% of male control value by those same exposure levels.
Gonad histopathology:
There were no treatment-related histopathologic changes either in the testes or ovaries of fathead minnows exposed to test substance at all concentrations tested. The pattern of predominant germ cell distribution (staging) in ovaries or testes did not show any meaningful differences between the controls and all test substance treated groups.
- Cumulative mortality at each concentration and for each recommended observation time if possible: was given under "Any other information on results including tables"
- Mortality in the controls: Four fish mortalities were observed in the control group.
- Behavioural observation of the fish: Given under "Any other information on results including tables"

1. Mean ± Standard Deviation Percent Mortality for P. promelas in different test substance treatment groups over the 21 Day study period (median values in parentheses)

Time Weighted Mean Measured test substance Concentration (μg/L)	Vessel #	% Mortality (Males)	% Mortality (Females)	% Mortality (Overall)
<0.0312 μg/L (water control)	3	0	0	0
	8	0	25	16.7
	10	0	25	16.7
	14	50	25	33.3
	Mean N^a=4	12.5 ± 25.00 (0)	18.8 ± 12.50 (25.0)	16.7 ± 13.60  (16.7)
0.251	4	0	25	16.7
	5	0	0	0
	11	0	0	0
	13	50	0	16.7
	Mean N^a=4	12.5 ± 25.00 (0)	6.3 ± 12.50 (0)	8.4 ± 9.64 (8.4)
0.812	1	50	25	33.3
	6	50	0	16.7
	12	0	0	0
	16	0	0	0
	Mean N^a=4	25.0 ± 28.87 (25.0)	6.3 ± 12.50 (0)	12.5 ± 15.95 (8.4)
3.02	2	0	0	0
	7	50	0	16.7
	9	0	25	16.7
	15	0	0	0
	Mean N^a=4	12.5 ± 25.00 (0)	6.3 ± 12.50 (0)	8.4 ± 9.64 (8.4)

a: Indicates number of replicate test vessels per treatment group, not total number of fish

Table 2: Abnormal behavior and appearance observations (including cumulative mortality) for P. promelas in different test substance treatment groups over the 21 Day study period.

Time Weighted Mean Measured test substance Concentration (μg/L)	Vessel #	Observations
<0.0312 μg/L (water control)	3	1F injured right eye; 1F injury on under belly
	8	1F skin discoloration(yellow), dead
	10	1F dead
	14	1M injured caudal fin, dead 1F lethargic, dead
0.251	4	1F dead
	5	1F vertical banding/dark coloration present
	11	1F bulging eye; 1F dark coloration present
	13	1F injured (bulging) eyes, scales missing 1M dead
0.812	1	1F bulging eye, eye missing, dead 1M dead
	6	1F bleeding mouth, side scrapes 1M loss of equilibrium, dead
	12	1M bent tail, thin, pale skin
	16	All Normal
3.02	2	All Normal
	7	1M injured eye, scales missing, dead 1M no dark coloration
	9	1F bloated, dead; 1F bloated; 1F side scrapes 1M no dark coloration
	15	1M injured/bloody snout, fat pad absent

Validity criteria fulfilled:

yes

Conclusions:

- At the end of the 21-day exposure, there were no statistically significant differences in mortality, wet weight or length among control and test substance-exposed fish up to the highest concentration of test substance tested, which was 3.02 μg/L.
- There were no statistically significant differences between control and test substance exposed fish in regards to the endpoints that were considered to be closely associated with perturbations to the HPG axis. Specifically, there were no statistically significant differences between control and test substance-exposed male and female fish in regards to gonado-somatic index, VTG, plasma concentrations of 17β estradiol and testosterone, and tubercle score. Furthermore, there were no significant treatment-related effects observed when ovaries and testes were examined histopathologically.
- The significant reductions in fecundity observed among test substance-exposed fish in the present study are most likely associated with inhibition of cholinesterase.

Executive summary:

The study was conducted according to guideline OECD 229 to evaluate the short-term reproduction assay using the fathead minnow, pimephales promelas. The test has pre-exposure phase for 15-day in flow-through conditions where water was given to fish. Following the pre-exposure phase, the test substance was exposed to fish for 21-days in flow-through condition. The nominal test concentrations were 0 (water control), 0.2, 0.64, and 2 μg/L. The time-weighted mean measured test concentrations are <LLQ (water control), 0.251, 0.812, and 3.02 μg/L.

Based on time-weighted mean measured concentrations, the results showed that there were eleven incidences of mortality, four in the controls, two in the 0.251 μg/L treatment group, three in the 0.812 μg/L treatment group and two in the 3.02 μg/L treatment group. There were no statistically significant differences in mortality among control and test substance exposed fish at the end of the study. Transient observations of abnormal behavior and appearance (i.e. discoloration, bent tail, loss of equilibrium, lethargy, and bloating), were observed in all treatment groups (including the control group), with no indication that test substance-exposed fish were experiencing overt toxicity. There were no statistically significant changes in wet weight or length among control and test substance-exposed fish and there were no statistically significant changes among control and test substance exposed fish in regards to the endpoints that are more closely associated with perturbations to the HPG axis such as gonadosomatic index, VTG, plasma concentrations of 17β-estradiol and testosterone, and tubercle score. Furthermore, there were no treatment-related histopathological effects in the ovaries and testes. The lack of treatment-related effects on this suite of endpoints indicate that test substance is not directly causing perturbations to the HPG axis of fish.

A statistically significant decrease in fecundity was noted among fish in all treatment levels of test substance at test termination. The fecundity response decreased in a dose dependent manner and therefore cholinesterase concentrations were analyzed in male and female fish. Female brain cholinesterase was statistically significantly reduced in a dose-dependent mann test substance er at all tested concentrations of test substance and male brain cholinesterase was statistically significantly reduced in the 0.812 and 3.02 μg/L treatment groups. At the lowest concentration of test substance tested in the present study (0.251 μg/L test substance), female brain cholinesterase was reduced to 60% of controls and male brain cholinesterase was reduced, though not statistically significant, to 65% of controls. The significant reductions in fecundity observed among test substance-exposed fish in the present study are most likely associated with inhibition of cholinesterase. Since brain cholinesterase activity was demonstrably reduced at the same concentrations of test substance that resulted in reduced fecundity, and since there were no alterations in more specific endpoints associated with HPG activity, namely secondary sex characteristics, concentrations of circulating sex steroids, VTG, gonado-somatic index, and gonadal histopathology, test substance is likely not active in the HPG axis of fish. Test substance likely exerts effects on fish reproduction via cholinesterase inhibition, the known mode of action for the organophosphate class of compounds.

Description of key information

Freshwater

21-day NOEC (RainbowTrout): 1.8 µg/L (Based on growth rate), 3.2 µg/L (Based on mortality); OECD 204, Reliability = 1

32 -day NOEC (Fatheand Minnow): 568 ng/L; EPA 72 -5, Reliability = 1

35 -day NOEC (Fathead Minnow): 1.10 -1.22 µg/L (highest dose tested); OECD 229, Reliability = 1

36 -day NOEC (Fathead Minnow): 3.02 µg/L (highest dose tested); OECD 229, OPPTS 890.1350, Reliability = 1

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

568 ng/L

Additional information

Several chronic fish tests were conducted on freshwater species. NOELs in freshwater species ranged from 568 ng/L to 3.2 µg/L.