Chlorothalonil

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 Sep 2013 to 7 Oct 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1350 (Mysid Chronic Toxicity Test)

Version / remarks:

1996

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples were removed from alternating replicate solutions of each treatment level and the control on days 0, 7, 14, 21 and 28 for analysis of the test substance concentration. The exposure system was also sampled on day 1 due to a sample processing error on day 0, and again on day 16 due to relatively low recoveries on day 14. The day 14 recoveries were inconsistent with previous analytical intervals. Samples of the diluter stock solution (saturator column effluent) were also analyzed at each of these intervals during the in-life phase of the definitive study.

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

STOCK SOLUTION PREPARATION
For this exposure, two glass wool saturator columns were used to deliver the test substance to the exposure system. The glass columns were packed with glass wool, and then coated with the test substance. The columns were designed to provide a constant flow of nearly saturated aqueous solution (0.6 mg/L) of the test substance to the diluter system without the use of a carrier solvent. Columns were constructed entirely of chemically inert materials.
To construct the columns, the glass housing was firmly packed with approximately 15% of the total column volume of glass wool. This provides ample surface area inside the column for the exposure substance to adhere once the column preparation is complete. After the column was packed, the end fittings were placed on the column.
To coat a column, approximately 1.6 g of the test substance was diluted with 75 mL of acetone. Approximately half of this solution was poured into the glass column. After all of the solution was added, the column was attached to a vacuum pump. The vacuum pump was used to draw the solution evenly throughout the column to uniformly coat the wool with the test substance and evaporate the remaining acetone. After it appeared that all of the wool was coated and all the solution appeared to be evaporated, the remaining solution was added to the column and the procedure was repeated. The column was then detached from the vacuum pump and attached to another pump, which delivered a flow of water through the column at a rate of 12.9 mL/min. After approximately two days of flushing, the column was attached to another pump at a rate of 5.0 mL/min or 0.0514 L/cycle. For this exposure, two columns were placed in series, saturator columns were prepared prior to exposure initiation and weekly thereafter. Each week, a new column was prepared and dilution water was delivered through the bottom of the first column. As the water exited the first column, it flowed directly through the second column and exited at the top into the mixing chamber. Saturator column trials performed prior to the range-finding exposure measured a stable and consistent dose of approximately 0.6 mg/L. This value was used to calculate the appropriate flow rate of stock solution into the diluter system.

EXPOSURE SYSTEM
Prior to exposure initiation, an deliver pump was calibrated to deliver 5.0 mL/min, for a total of 0.052 L/cycle of the saturated solution to the diluter system's mixing chamber. The mixing chamber was positioned over a water-driven magnetic stir plate and was partially submerged within an ultrasonic water bath. The continuous stirring and sonication aided in the mixing of the column effluent with dilution water. The solution contained in the mixing chamber constituted the highest nominal test concentration (16 µg/L) and was subsequently proportionally diluted (50%) to provide the remaining nominal exposure concentrations (8.0, 4.0, 2.0, 1.0 and 0.50 µg/L). A set of negative control vessels was also established which contained the same dilution water and was maintained under the same conditions as the treatment level vessels, but contained no the test substance.

Test organisms (species):

Americamysis bahia (previous name: Mysidopsis bahia)

Details on test organisms:

TEST ORGANISM
- Common name: Saltwater mysids
- Age at study initiation: ≤ 22.5 hours old
- Adult, gravid female mysids were isolated from the brood stock the day prior to initiation of the exposure and held in isolation. The exposure was then initiated with young collected from the isolated females.
- The brood stock was maintained in house for approximately 21 months prior to use.

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions: Mysids were cultured in six recirculating 80 L glass aquaria containing dilute, natural seawater. Standard aquarium undergravel filters were used to provide aeration and a current conducive to feeding.
- Brood stock and test organisms were cultured and tested in seawater from the same source.
- Salinity: 21 - 23‰
- Dissolved oxygen: 90.0 - 90.7% of saturation
- pH: 7.8 - 7.9
- Temperature: 25 - 26 °C (using a commercial aquarium heater )
- Photoperiod: 16 hours light/ 8 hours dark
- Light intensity: 52 - 69 footcandles

FEEDING
- Feed type and frequency: Throughout the test, mysids were fed live brine shrimp (Artemia salina) nauplii, ≤ 48 hours old (post-hydration), twice daily. At least one of these feedings was with brine shrimp nauplii enriched with a substance high in saturated fatty acids.
- Feeding amount: Densities per mL of concentrated collections of nauplii from Artemia cultures created from daily hydrated cysts were determined by triplicate counts of aliquots pipetted onto filter paper. Based on these densities, various volumes of nauplii were fed to mysids per feeding in accordance with the following regime:ations for F0 retention chambers (containing 20 mysids each) were approximately 90 nauplii/mysid on test days 0 to 3, 135 nauplii/mysid on test days 4 to 6, 180 nauplii/mysid on test days 7 to 9 and 225 nauplii/mysid on test day 10 to pairing. From day of pairing until test termination, the F0 pairing chambers were fed approximately 450 nauplii/mysid and the retention chambers were fed approximately 3600 nauplii/chamber. After F1 chambers were initiated, chambers were fed a ration of approximately 90 nauplii/mysid.
- All treatments and the control received, as near as reasonably possible, the same ration of food. Excess brine shrimp and organic debris in the test chambers was removed prior to the addition of new nauplii.
- Feeding quality: Prior to use of a new batch of feed, a representative sample of the food source was analyzed for the presence of pesticides, PCBs, and toxic metals None of these compounds were detected at concentrations that are considered toxic in any of the food samples analyzed in agreement with ASTM (2002) standard practice. Based on the analysis for pesticides and the performance of the control organisms, the food source was considered to be of acceptable quality since all analyte concentrations were below levels of concern and control organisms exceeded guideline criteria.

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

28 d

Test temperature:

26 - 27 ºC

pH:

7.9 - 8.2

Dissolved oxygen:

- 5.09 - 6.88 O2 mg/L
- 71.0 - 96.3%

Salinity:

19 - 21‰

Nominal and measured concentrations:

- Nominal concentration: 0 (Control), 0.50, 1.0, 2.0, 4.0, 8.0 and 16 µg/L.
- Time-weighted average concentration: NA (Control), 0.40, 0.87, 1.7, 3.8, 6.4 and 14 µg/L, respectively (NA = Not Applicable). See Table 1 in 'Any other information on materials and methods incl. tables'.

Details on test conditions:

TEST SYSTEM
- Test vessel: Aquarium
- Vessel size and fill volume: 30 × 15 × 20 cm with a 10 cm high side drain that maintained a constant exposure solution volume of approximately 4.5 L.
- Type of flow-through: Proportional diluter
- Flow rate: Approximately 7.7 aquarium volume additions per day to provide a 90% test solution replacement rate of approximately 7 hours (The function of the diluter system (e.g., dilution water and saturator column output flow rates) was monitored daily and a complete check was performed twice each day. In addition, weekly analysis of the exposure solutions was used to verify proper operation of the diluter system.)
- Biomass loading: ≤ 0.003 g/L flowing solution per day or ≤ 0.020 g/L of solution at any time, in any replicate exposure aquarium during the 28-day exposure period.
- The exposure system was in proper operation for five days prior to exposure initiation to allow equilibration of the test substance in the diluter apparatus and exposure vessels.
- No. of vessels per concentration: 4
- No. of vessels per dilution water control: 4

F0 life-cycle exposure initiation
- For the first 11 days of exposure, each exposure aquarium contained one retention chamber. The retention chambers, used to retain sexually immature mysids, were constructed of glass petri dishes, 10 cm in diameter, 2 cm deep, to which a 14 cm high screen collar (350 µm mesh size opening) was attached with silicone sealant. The solution volume within each retention chamber was approximately 785 mL.
- No. organisms per retention chamber: 20
- Each exposure aquarium contained one retention chamber, yielding 20 mysids per replicate vessel and 80 organisms for each treatment level and the control.
- The exposure was initiated when the retention chambers were placed in their respective exposure aquaria.

F0 Life-Cycle Mysid Pairing
- Once all mysids appeared to be sexually mature (test day 12), male and female pairs were transferred to separate pairing chambers. Following this distribution, each exposure aquarium contained one retention chamber and a maximum of five pairing chambers. Pairing chambers, used to retain sexually mature male and female organisms, were constructed of 6 cm diameter petri dishes, to which a 14 cm high screen collar (350 µm mesh size opening) was attached with silicone sealant. Solution volume within each pairing chamber was approximately 250 mL.
- No. organisms per pairing chamber: 2 (one mature male and one mature female were randomly assigned)
- The length of time for brood appearance (i.e., gravid females) was noted for all first generation mysids prior to pairing (approximately day 11).
- Unpaired mysids were pooled and maintained in one of the initial retention chambers until they were paired or until test termination. When six or fewer unpaired mysids remained in a retention chamber, they were transferred to a separate pairing chamber in order to reduce the surface area of chambers within the exposure vessel. Male mysids from this pool were used to replace dead males from the paired (male/female) groups. Females that died in the pairing chambers were replaced at the Study Director’s discretion. For example, if a female within a pairing chamber died prior to the onset of reproduction, that female may be replaced. Once reproduction within the study had begun, females were not replaced.

F1 Generation
- During the reproductive phase, offsprings (F1 mysids) were removed from mysid chambers in each replicate vessel, pooled and placed in a separate pairing chamber in that replicate. Chambers were established based on the number of available juvenile F1 mysids; therefore, each chamber was not necessarily initiated on the same day. One F1 group was established and monitored for each replicate vessel.
- No. organisms per replicate: 10 offsprings (if 10 offspring could not be collected, a reduced number of young was collected and evaluated;
n > 5)
- The chambers with F1 mysids were established to monitor survival for approximately 96 hours post-release. This observation period ensured an equal observation period across all treatment levels and the control prior to exposure termination.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilute, filtered, natural seawater was used as dilution and control water during this study. Seawater was collected from the Cape Cod Canal, Bourne, Massachusetts from about 1 to 4 meters offshore at a depth of approximately 0.5 meters. The seawater was then transferred by a pump (fiberglass reinforced thermoplastic housing) through polyvinyl chloride (PVC) pipes and transported to the laboratory in a 3400 L fiberglass holding tank. In the laboratory, the seawater was adjusted to a salinity of 20 ± 3‰ with laboratory well water, and filtered through 20, 5 µm and 1 µm polypropylene core filters prior to use. The seawater was pumped under constant pressure through PVC pipes to the intermittent-flow proportional diluter system.
- Routine analyses were conducted periodically on representative samples of the water source for the presence of pesticides, PCBs, and toxic metals. None of these compounds were detected at concentrations that are considered toxic in any of the water samples analyzed in agreement with ASTM (2002) standard practice. Based on these analyses and the performance of the control organisms, the water source was considered to be of acceptable quality since all analyte concentrations were below levels of concern and control organisms exceeded guideline performance criteria.
- Representative samples of the dilution water were analyzed monthly for total organic carbon (TOC) concentration. The TOC concentration of the dilution water was 1.1 and 1.2 mg/L for September and October 2013, respectively.
- Intervals of water quality measurement: Temperature, dissolved oxygen concentration, pH and salinity were measured in each replicate
on day 0 and alternated between replicates daily thereafter throughout the exposure period, for each treatment level and the control. In addition, exposure solution temperature was continuously monitored in one control vessel (replicate A).

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with a 30 minutes transition period
- Light intensity: 24 to 43 footcandles (260 to 460 lux)

EFFECT PARAMETERS MEASURED:
- Mysids counting and biological observation: In order to observe the mysids during the exposure period, each retention chamber was gently lifted from the aquaria daily. The chamber was then placed into a dish containing dilution water and the dish was placed on a light table. During this procedure, the water level in each chamber was reduced by allowing water to drain through the screen. The numbers of dead and living organisms were counted and any abnormal appearance or behavior was recorded. Due to the rapid movement of mysids in a single chamber containing up to 20 mysids, survival of the test organisms was estimated for the first 11 days of the test, i.e., prior to pairing of the mysids. After males and females had been paired (day 12) actual counts of survival were made and the number of dead males and females, the number of offspring produced by each individual female and any abnormal appearance or behavior was recorded. Observations were made daily throughout the study. Dead parental mysids and offspring were recorded, removed, and discarded when observed during the test. Mortality was defined as lack of movement after gentle prodding with a glass pipette. Following the observation period, retention chambers were brushed clean, debris was removed, and they were returned to the respective aquaria.
- F1 generation biological observations and survival: At the time an F1 generation pairing chamber was established and daily thereafter for 96 hours, observations of stress, abnormal behavior (including discoloration, immobilization and inability to maintain position in the water column), and survival were made. Dead mysids (defined as the absence of mobility and failure to respond to gentle prodding) were recorded and removed from each replicate test vessel daily. Missing mysids were considered dead.
- Mean body length and dry weight determinations: At test termination, all mysids were euthanized by immersion in ice-cold, deionized water. The mysids were carefully removed, blotted dry on absorbent paper, and separated into male and female groups for each replicate exposure level. A digital photograph was then taken of each mysid using a binocular dissection microscope for individual body length measurements. Individual body length was measured to the nearest 0.01 mm. Following these procedures, male and female mysids were transferred to aluminum pans, dried in an oven at 96 to 100 °C for approximately 22 hours and then placed in a desiccator. Individual dry body weight to the nearest 0.01 mg was determined using an analytical balance. Individual lengths and weights of all surviving males and females were recorded separately for each replicate of each concentration and the control.
- Reproductive success: Reproductive success was calculated for each replicate aquarium (treatments and the control) as the total number of offspring produced per female. In addition, the percentage of actively reproducing females in each replicate of each treatment and the control was determined.

RANGE FINDING SYUDY
- A preliminary 28-day flow-through exposure was conducted at the test facility on juvenile mysids (40 mysids per treatment level; 20 per replicate). The procedures used for this exposure were similar to the procedures used for the definitive exposure.
- Nominal concentrations: Control, 0.25, 0.50, 1.0, 2.0 and 4.0 µg/L
- Results used to determine the conditions for the definitive study: F1 generation 96-hour post-release survival among mysids exposed to the 0.25, 1.0, 2.0 and
4.0 µg/L treatment levels averaged 95, 95, 95 and 90%, respectively. F1 generation 96-hour post-release survival in the control and the 0.50 µg/L treatment level averaged 100%. Based on these results and consultation with the Study Sponsor, the nominal concentrations selected for the definitive exposure were 0.50, 1.0, 2.0, 4.0, 8.0 and 16 µg/L.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.4 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Mean number of offspring per female organisms

Details on results:

An overview of the results is provided in Table 2 and Table 3 in 'Any other information on results incl. tables'.
At test termination, the mysids in the control exceeded the performance criteria of the OCSPP 850.1350 draft guideline and the test protocol (≥ 70% survival of F0 mysids between pairing and exposure termination, ≥ 75% of the females in the control released young and the control organisms produced ≥ 8 offspring per female). Post-pairing survival for the control mysids ranged from 81 to 100% for all replicates; the control mean was 90%. The mean percentage of reproductively active females for the control mysids was 100%. The reproduction for the control mysids ranged from 18.0 to 24.0 offspring per female for all replicates; the control mean was 21.7 offspring per female. No behavioral abnormalities were observed during the exposure period.
The calculation of male and female survival began following pairing; any male or female deaths which occurred prior to pairing are not included in this calculation.
At test termination, mean survival of 87% was observed among male mysids in the control. Mean survival of 85, 81, 83, 76, 80 and 70% was observed among male mysids exposed to the 0.40, 0.87, 1.7, 3.8, 6.4 and 14 µg/L treatment levels, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant difference in male survival among organisms exposed to any of the treatment levels tested compared to the control data. Therefore, the NOEC for this endpoint was determined to be 14 µg/L and the LOEC could not be determined.
At test termination, mean survival of 93% was observed among female mysids in the control. Mean survival of 83, 90, 94, 85, 91 and 95% was observed among female mysids exposed to the 0.40, 0.87, 1.7, 3.8, 6.4 and 14 µg/L treatment levels, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant difference in female survival among organisms exposed to any of the treatment levels tested compared to the control data. Therefore, the NOEC for this endpoint was determined to be 14 and the LOEC could not be determined.
Following 28-days of exposure, mean 28-day survival of 83% was observed among organisms in the control. Mean 28-day survival of 72, 79, 81, 71, 72 and 66% was observed among mysids exposed to the 0.40, 0.87, 1.7, 3.8, 6.4 and 14 µg/L treatment levels, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant difference in survival among organisms exposed to any of the treatment levels tested compared to the control data. Therefore, the NOEC for this endpoint was determined to be 14 µg/L and the LOEC could not be determined.
At test termination, the mean number of offspring per female for organisms in the control was 21.7. The mean number of offspring per female was 17.5, 17.0, 15.0, 16.8, 17.5 and 16.9 among mysids exposed to the 0.40, 0.87, 1.7, 3.8, 6.4 and 14 µg/L treatment levels, respectively. Dunnett’s Multiple Comparison Test determined a significant difference in the mean number of offspring per female organisms exposed to the 0.87, 1.7, 3.8 and 14 µg/L treatment levels tested compared to the control data. Therefore, the NOEC and LOEC for this endpoint were determined to be 0.40 and 0.87 µg/L, respectively

Reported statistics and error estimates:

See statistical analysis in "Any other information on materials and methods incl. tables"

Table 2. Effects of the test substance on the survival and reproductive success of mysids (Americamysis bahia). The numbers in brackets indicate the standard deviation.

Time-weighted average concentration (µg ai/L)	Male survival (%)	Female survival (%)	Post pairing survival (%)	28-day survival (%)	No. of offspring per female	F1 survival at 96 hours post release (%)
Dilution water control	87 (9)	93 (8)	90 (8)	83 (12)	21.7 (2.8)	98 (5)
0.40	85 (11)	83 (13)	83 (9)	72 (16)	17.5 (2.4)	98 (5)
0.87	81 (22)	90 (11)	86 (11)	79 (12)	17.0* (1.7)	93 (5)
1.7	83 (22)	94 (7)	90 (11)	81 (12)	15.0* (3.2)	95 (10)
3.8	76 (22)	85 (7)	80 (14)	71 (18)	16.8* (1.3)	98 (5)
6.4	80 (18)	91 (13)	84 (11)	72 (13)	17.5 (1.5)	98 (5)
14	70 (25)	95 (10)	79 (19)	66 (18)	16.9* (3.8)	100 (0)

Table 3. Effects of the test substance on the growth of mysids (Americamysis bahia) following exposure for 28 days in a flow-through test.

Time-weighted average concentration (µg ai/L)	Mean total body length (mm)		Mean dry body weight (mg)
	Males	Females	Males	Females
Dilution water control	7.20 (0.15)	7.42 (0.18)	0.84 (0.06)	1.22 (0.10)
0.40	7.07 (0.08)	7.35 (0.20)	0.81 (0.03)	1.19 (0.09)
0.87	7.22 (0.14)	7.32 (0.08)	0.88 (0.03)	1.18 (0.07)
1.7	6.94 (0.19)	7.28 (0.23)	0.80 (0.02)	1.04* (0.04)
3.8	6.81* (0.23)	7.31 (0.41)	0.85 (0.05)	1.19 (0.12)
6.4	7.04 (0.18)	7.33 (0.12)	0.89 (0.05)	1.22 (0.04)
14	6.97 (0.09)	7.24 (0.24)	0.87 (0.02)	1.07 (0.15)

* Significantly reduced compared to the control, based on Dunnett’s Multiple Comparison Test. However due to the lack of statistical significance at higher treatment levels tested, the effect observed at this treatment level was not considered to be treatment-related since a clear dose-response relationship was not observed.

Validity criteria fulfilled:

not specified

Conclusions:

Based on the findings, the 28-day NOEC for the mean number of offspring per female organisms was determined to be 0.40 µg/L.

Executive summary:

This study was performed to determine the chronic (full life-cycle) toxicity of the test substance to the mysid, Americamysis bahia, under flow-through conditions. This 28-day study was conducted according to EPA 850.1350 guideline and in compliance with GLP criteria. The life-cycle toxicity test was designed to provide six concentrations of the test substance and a dilution water control to four replicate aquaria. The test chambers were impartially positioned within a water bath to maintain temperature. Four replicate tanks were prepared for the controls and each test solution. Twenty mysids were randomly allocated to each prepared test vessel. The mysides were exposed to5 µm filtered, salinity 19 - 21‰ seawater withthe test substance nominal concentrations of 0.50, 1.0, 2.0, 4.0, 8.0 and 16 µg/L. The time-weighted average were 0.40, 0.87, 1.7, 3.8, 6.4 and 14 µg/L (measured by GC) (a constant exposure solution volume of approximately 4.5 L). The test area was illuminated at an intensity range of 24 to 43 footcandles (260 to 460 lux), measured during the in-life exposure, and a photoperiod of 16 hours light and 8 hours darkness with a 30-minute transition period. The study was conducted in a water bath designed to maintain the test solution temperatures at 25 ± 2 °C. Temperature, dissolved oxygen concentration, pH and salinity were measured in each replicate on day 0 and alternated between replicates daily thereafter throughout the exposure period, for each treatment level and the control. In addition, exposure solution temperature was continuously monitored in one control vessel.

At test termination, male and female mysids were transferred to aluminium pans, dried in an oven at 96 to 100 °C for approximately 22 hours and then placed in a desiccator. Individual dry body weight to the nearest 0.01 mg was determined using an analytical balance. Individual lengths and weights of all surviving males and females were recorded separately for each replicate of each concentration and the control. Reproductive success was calculated for each replicate aquarium (treatments and the control) as the total number of offspring produced per female. In addition, the percentage of actively reproducing females in each replicate of each treatment and the control was determined. Observations were made daily throughout the study. Dead parental mysids and offspring were recorded, removed, and discarded when observed during the test. Mortality was defined as lack of movement after gentle prodding with a glass pipette. Following the observation period, retention chambers were brushed clean, debris was removed, and they were returned to the respective aquaria.

At test termination, the mysids in the control exceeded the performance criteria of the OCSPP 850.1350 draft guideline and the test protocol (≥ 70% survival of F0 mysids between pairing and exposure termination, ≥ 75% of the females in the control released young and the control organisms produced ≥ 8 offspring per female). Post-pairing survival for the control mysids ranged from 81 to 100% for all replicates; the control mean was 90%. The mean percentage of reproductively active females for the control mysids was 100%. The reproduction for the control mysids ranged from 18.0 to 24.0 offspring per female for all replicates; the control mean was 21.7 offspring per female. No behavioural abnormalities were observed during the exposure period.

The calculation of male and female survival began following pairing; any male or female deaths which occurred prior to pairing are not included in this calculation. At test termination, mean survival of 87% was observed among male mysids in the control. Mean survival of 85, 81, 83, 76, 80 and 70% was observed among male mysids exposed to the 0.40, 0.87, 1.7, 3.8, 6.4 and 14 µg/L treatment levels, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant difference in male survival among organisms exposed to any of the treatment levels tested compared to the control data. Therefore, the NOEC for this endpoint was determined to be 14 µg/L and the LOEC could not be determined.

At test termination, mean survival of 93% was observed among female mysids in the control. Mean survival of 83, 90, 94, 85, 91 and 95% was observed among female mysids exposed to the 0.40, 0.87, 1.7, 3.8, 6.4 and 14 µg/L treatment levels, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant difference in female survival among organisms exposed to any of the treatment levels tested compared to the control data. Therefore, the NOEC for this endpoint was determined to be 14 and the LOEC could not be determined.

Following 28-days of exposure, mean 28-day survival of 83% was observed among organisms in the control. Mean 28-day survival of 72, 79, 81, 71, 72 and 66% was observed among mysids exposed to the 0.40, 0.87, 1.7, 3.8, 6.4 and 14 µg/L treatment levels, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant difference in survival among organisms exposed to any of the treatment levels tested compared to the control data. Therefore, the NOEC for this endpoint was determined to be 14 µg/L and the LOEC could not be determined.

At test termination, the mean number of offspring per female for organisms in the control was 21.7. The mean number of offspring per female was 17.5, 17.0, 15.0, 16.8, 17.5 and 16.9 among mysids exposed to the 0.40, 0.87, 1.7, 3.8, 6.4 and 14 µg/L treatment levels, respectively. Dunnett’s Multiple Comparison Test determined a significant difference in the mean number of offspring per female organisms exposed to the 0.87, 1.7, 3.8 and 14 µg/L treatment levels tested compared to the control data.

Based on the findings and time-weighted average concentrations of the test substance to the mean number of offspring per female organism endpoint, the 28-day NOEC was determined to be 0.40 µg/L.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

3 Aug 1981 to 14 Sep 1981

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EPA guidelines for registering pesticides in the United States, specifically Subpart E, Hazard Evaluation: Wildlife and Aquatic Organisms

Version / remarks:

1980

Deviations:

yes

Remarks:

Test solutions were delivered to the aquaria at a rate of five aquarium volumes per day instead of the protocol recommended four.

GLP compliance:

yes

Remarks:

draft

Test organisms (species):

Daphnia magna

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

42 d

Remarks on exposure duration:

F0 21 days exposure to the test substance followed by a second generation (F1) exposure under similar conditions for an additional 21 days.

Key result

Duration:

42 d

Dose descriptor:

NOEC

Effect conc.:

35 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

F0

Results:

Actual concentrations were 54 - 79% of nominal during the chronic test. Before initiating the test a pretest was performed (without daphnids): actual concentrations were comparable to those with daphnids. Mean measured concentrations were 0.0038, 0.0065, 0.016, 0.035, and 0.079 mg/L. Only in the highest dose group significant dose-related mortality occurred. The reproduction (F0) per female was only reduced at the highest concentration. The offspring of the second generation F1 was higher at all test levels compared to the control, in particular in the highest dose level (but these daphnids were taken from the F0 control group). The age (< 72 hours old) might be the cause for this discrepancy.

Validity criteria fulfilled:

not specified

Conclusions:

Based on the findings, the NOEC was determined to be 35 µg/L.

Executive summary:

The chronic effects of the test substance (98.8%) on the water flea (Daphnia magna) was studied under a flow through conditions at the measured concentrations of 3.8, 6.5, 16, 35 and 79 µg/L (measured by GC, nominal concentrations of 6.2, 12, 25, 50 and 100 µg/L, respectively). In addition, both negative control and solvent control (acetone, max. 0.5 mL/L) were included in this study. This 42-day study was performed according to EPA guidance for registering pesticides in the United States, specifically Subpart E, Hazard Evaluation: Wildlife and Aquatic Organisms. It was compliant with GLP criteria (draft). The study used five daphnids per vessel and four vessels per concentration. The exposure lasted 21 days per generation. Daphnids (< 24 hours old) were used at start and fed daily. Their offspring from days 20 and 21 (< 72 hours old) were used to initiate the second generation. Since only one daphnid survived the highest concentration, control offspring was used for the highest level.

Only in the highest dose group significant dose-related mortality occurred. The reproduction (F0) per female was only reduced at the highest concentration (79 µg/L). The offspring of the second generation F1 was higher at all test levels compared to the control, in particular in the highest dose level (but these daphnids were taken from the F0 control group). The age (< 72 hours old) might be the cause for this discrepancy. Based on the reproduction of F0, the NOEC is 35 µg/L.

Description of key information

Freshwater, 42-d NOEC = 35 µg/L, Daphnia magna, reproduction, EPA guidance for registering pesticides in the U.S., Shults 1982.

Marine water, 28-d NOEC = 0.40 µg/L, Americamysis bahia, reproduction (mean number of offspring per female organism), EPA TG 850.1350, Schwader 2014.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

35 µg/L

Marine water invertebrates

Marine water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.4 µg/L

Additional information

Freshwater

The chronic effects of the test substance (98.8%) on the water flea (Daphnia magna) was studied under a flow through conditions at the measured concentrations of 3.8, 6.5, 16, 35 and 79 µg/L (measured by GC, nominal concentrations of 6.2, 12, 25, 50 and 100 µg/L, respectively). In addition, both negative control and solvent control (acetone, max. 0.5 mL/L) were included in this study. This 42-day study was performed according to EPA guidance for registering pesticides in the United States, specifically Subpart E, Hazard Evaluation: Wildlife and Aquatic Organisms. It was compliant with GLP criteria (draft). The study used five daphnids per vessel and four vessels per concentration. The exposure lasted 21 days per generation. Daphnids (< 24 hours old) were used at start and fed daily. Their offspring from days 20 and 21 (< 72 hours old) were used to initiate the second generation. Since only one daphnid survived the highest concentration, control offspring was used for the highest level.

Only in the highest dose group significant dose-related mortality occurred. The reproduction (F0) per female was only reduced at the highest concentration (79 µg/L). The offspring of the second generation F1 was higher at all test levels compared to the control, in particular in the highest dose level (but these daphnids were taken from the F0 control group). The age (< 72 hours old) might be the cause for this discrepancy. Based on the reproduction of F0, the NOEC is 35 µg/L.

Marine water

This study was performed to determine the chronic (full life-cycle) toxicity of the test substance to the mysid, Americamysis bahia, under flow-through conditions. This 28-day study was conducted according to EPA 850.1350 guideline and in compliance with GLP criteria. The life-cycle toxicity test was designed to provide six concentrations of the test substance and a dilution water control to four replicate aquaria. The test chambers were impartially positioned within a water bath to maintain temperature. Four replicate tanks were prepared for the controls and each test solution. Twenty mysids were randomly allocated to each prepared test vessel. The mysides were exposed to 5 µm filtered, salinity 19 - 21‰ seawater with the test substance nominal concentrations of 0.50, 1.0, 2.0, 4.0, 8.0 and 16 µg/L. The time-weighted average were 0.40, 0.87, 1.7, 3.8, 6.4 and 14 µg/L (measured by GC) (a constant exposure solution volume of approximately 4.5 L). The test area was illuminated at an intensity range of 24 to 43 footcandles (260 to 460 lux), measured during the in-life exposure, and a photoperiod of 16 hours light and 8 hours darkness with a 30-minute transition period. The study was conducted in a water bath designed to maintain the test solution temperatures at 25 ± 2 °C. Temperature, dissolved oxygen concentration, pH and salinity were measured in each replicate on day 0 and alternated between replicates daily thereafter throughout the exposure period, for each treatment level and the control. In addition, exposure solution temperature was continuously monitored in one control vessel.

At test termination, male and female mysids were transferred to aluminium pans, dried in an oven at 96 to 100 °C for approximately 22 hours and then placed in a desiccator. Individual dry body weight to the nearest 0.01 mg was determined using an analytical balance. Individual lengths and weights of all surviving males and females were recorded separately for each replicate of each concentration and the control. Reproductive success was calculated for each replicate aquarium (treatments and the control) as the total number of offspring produced per female. In addition, the percentage of actively reproducing females in each replicate of each treatment and the control was determined. Observations were made daily throughout the study. Dead parental mysids and offspring were recorded, removed, and discarded when observed during the test. Mortality was defined as lack of movement after gentle prodding with a glass pipette. Following the observation period, retention chambers were brushed clean, debris was removed, and they were returned to the respective aquaria.

At test termination, the mysids in the control exceeded the performance criteria of the OCSPP 850.1350 draft guideline and the test protocol (≥ 70% survival of F0 mysids between pairing and exposure termination, ≥ 75% of the females in the control released young and the control organisms produced ≥ 8 offspring per female). Post-pairing survival for the control mysids ranged from 81 to 100% for all replicates; the control mean was 90%. The mean percentage of reproductively active females for the control mysids was 100%. The reproduction for the control mysids ranged from 18.0 to 24.0 offspring per female for all replicates; the control mean was 21.7 offspring per female. No behavioural abnormalities were observed during the exposure period.

The calculation of male and female survival began following pairing; any male or female deaths which occurred prior to pairing are not included in this calculation. At test termination, mean survival of 87% was observed among male mysids in the control. Mean survival of 85, 81, 83, 76, 80 and 70% was observed among male mysids exposed to the 0.40, 0.87, 1.7, 3.8, 6.4 and 14 µg/L treatment levels, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant difference in male survival among organisms exposed to any of the treatment levels tested compared to the control data. Therefore, the NOEC for this endpoint was determined to be 14 µg/L and the LOEC could not be determined.

At test termination, mean survival of 93% was observed among female mysids in the control. Mean survival of 83, 90, 94, 85, 91 and 95% was observed among female mysids exposed to the 0.40, 0.87, 1.7, 3.8, 6.4 and 14 µg/L treatment levels, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant difference in female survival among organisms exposed to any of the treatment levels tested compared to the control data. Therefore, the NOEC for this endpoint was determined to be 14 and the LOEC could not be determined.

Following 28-days of exposure, mean 28-day survival of 83% was observed among organisms in the control. Mean 28-day survival of 72, 79, 81, 71, 72 and 66% was observed among mysids exposed to the 0.40, 0.87, 1.7, 3.8, 6.4 and 14 µg/L treatment levels, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant difference in survival among organisms exposed to any of the treatment levels tested compared to the control data. Therefore, the NOEC for this endpoint was determined to be 14 µg/L and the LOEC could not be determined.

At test termination, the mean number of offspring per female for organisms in the control was 21.7. The mean number of offspring per female was 17.5, 17.0, 15.0, 16.8, 17.5 and 16.9 among mysids exposed to the 0.40, 0.87, 1.7, 3.8, 6.4 and 14 µg/L treatment levels, respectively. Dunnett’s Multiple Comparison Test determined a significant difference in the mean number of offspring per female organisms exposed to the 0.87, 1.7, 3.8 and 14 µg/L treatment levels tested compared to the control data.

Based on the findings and time-weighted average concentrations of the test substance to the mean number of offspring per female organism endpoint, the 28-day NOEC was determined to be 0.40 µg/L.