1-[(4-chlorophenyl)methyl]-1-cyclopentyl-3-phenylurea

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-09-28 to 2002-08-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 72-4 (Fish Early Life-Stage and Aquatic Invertebrate Life-Cycle Studies)

Version / remarks:

1982

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ASTME 1241-92

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

1992

Deviations:

no

Principles of method if other than guideline:

There was a deviation from the study protocol between study day 5 and study day 9. Within this period problems occured with the syringe pump system in the highest test level at 345.1 µg a.s./L (Table 10). The pump was cleaned on day 6, but dose verifications on day 7 and 8 indicated a malfunction of the dosing system. So, the pump was replaced by a new calibrated pump on day 9. This deviation was only of short duration (4 days = 4% of the whole exposure period) within the egg stage of fish and did therefore not influence the results of the study negatively.

GLP compliance:

yes

Specific details on test material used for the study:

The test material is a colourless crystal.

Analytical monitoring:

yes

Details on sampling:

Samples of test solution, including controls, were taken from alternating replicate test chambers on days - 2, -1, 0, 7, 14, 21, 28, 35, 43, 49, 56, 63, 70, 77, 84, 91 and 94 to measure actual test substance exposure concentrations. The analysis was by HPLC.

Vehicle:

yes

Remarks:

Acetone (100 µL acetone per liter dilution water)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Controls: Dilution water control and solvent control.
- Chemical name of vehicle: Acetone
- Concentration of vehicle in test medium: 100 µL acetone per liter dilution water.
- Other relevant information: A total of 135 L/h dilution water was used in this study. This dilution water was divided into nine separate water streams of 15 L/h, controlled by flow meters. A diluter system with Prominent micro g/5 pumps was used for the periodical introduction of 1.5 mL/h of stock solutions with different concentrations of test substance. Stock solutions of test substance were prepared with acetone, to be added to each of these streams of dilution water (resulting in a solvent concentration of 100 µL acetone per liter of dilution water). This was not added to the dilution water control group or to the fertilization control group. The solvent control was pure acetone. All stock solutions were continuously stirred during the test by magnetic stirrers. Flow-splitting cells divided the water streams after the introduction of the stock solution and after passing through mixing chambers into four aliquots per test concentration before being delivered to replicate test chambers. Six nominal test substance concentrations of 9.4, 18.8, 37.5, 75.0, 150.0 and 300.0 µg a.s./L (ppb) were tested.
Eggs were incubated in incubation cups constructed from 8 cm diameter teflon pipes with stainless steel plates perforated with holes (hole diameter: 1.8 mm) on the bottom. These incubation cups were suspended in each replicate test chamber. To facilitate water circulation and keep the eggs clean, incubation cups were oscillated vertically (2 times per minute) with a lifting movement of approximately 4 cm in the test chamber by means of a rocker arm apparatus driven by a low rpm electric motor. The glass aquaria were approximately 12 cm x 14 cm with a water depth of 21 cm, yielding an approximate chamber volume of 3.5 liters and resulting in approximate 24 changes of test water per day.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout (Oncorhynchus mykiss)

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish: The eggs (~ 3000) and milt used in the study were collected from 3 female and 3 male adult brood fish.
- Method of collection of fertilised eggs: The unfertilized eggs and milt were acclimated from transport temperature to the temperature of 10°C over a period of 10 minutes. Upon acclimation, eggs were transferred to a dry stainless steel bowl and the milt was mixed with the eggs (with an autoclaved goose feather) for about 3 minutes. Dilution water (10° ± 1°C) was added until the eggs were covered. The eggs and milt were gently stirred for about 1 minutes to facilitate fertilization. After stirring, the eggs rested for about 5 minutes and were then gently rinsed with 10° + 1°C dilution water until the milt was no longer visible. The eggs were allowed to harden in water for approximately 1-2 hours before being randomly distributed into incubation cups.

Test type:

flow-through

Water media type:

other: Reconstituted water

Limit test:

no

Total exposure duration:

94 d

Hardness:

Mean: 2.7°dH (Range: 2.4 - 3.0°dH)
(1°dH = 17.8 mg/L CaCO3)

Test temperature:

Mean: 10.0°C (Range: 9.4 - 10.8°C)

pH:

Mean: 7.2 - 7.3 (Range: 7.0 - 7.4)

Dissolved oxygen:

Mean: 97 - 98% (Range: 92 - 101%)

Conductivity:

Mean: 119.1 µS/cm (Range: 100.8 - 130.9 µS/cm)

Nominal and measured concentrations:

Nominal test concentrations of 9.46 (9.40), 18.9 (18.8), 37.7 (37.5), 75.5 (75.0), 150.9 (150.0), and 301.8 (300.0) µg a.s./L (ppb).
Mean measured concentration of 10.2, 21.7, 40.0, 83.2, 167.4 and 345.1 µg a.s./L (ppb).

Details on test conditions:

TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): Eggs were incubated in incubation cups constructed from 8 cm diameter teflon pipes with stainless steel plates perforated with holes (hole diameter: 1.8 mm) on the bottom.
- Test vessel: glass aquaria
- Material, size, headspace, fill volume: The glass aquaria were approximately 12 cm x 14 cm with a water depth of 21 cm, yielding an approximate chamber volume of 3.5 liters
- Renewal rate of test solution (frequency/flow rate): 24 changes of test water per day.
- Aeration: no
- No. of fertilized eggs/embryos per vessel: 35 eggs at start of the test; 15 hatched individuals from day 43.
- No. of vessels per concentration (replicates): 4 replicate exposure chambers per concentration.
- No. of vessels per control (replicates): 4 replicates
- No. of vessels per vehicle control (replicates): 4 replicates
- Biomass loading rate: The biomass loading factor for the study was determined using the wet weights of the control and solvent control fish at the study termination (Table 6). The mean wet weight was 560.2 mg. The biomass loading factor based upon the 3.5 liter volume of a single growth chambers is 2401 mg per liter. The biomass loading factor based on a flow of 84 liters per day through each single test chamber, was 29 mg per liter and day. These values were well within the requirements to ensure adequate dissolved oxygen levels and to avoid fish crowding.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water was prepared by adding salt stock solutions to demineralized water (conductivity < 0.2 µS/cm) to yield the following nominal ionic concentrations (according to ISO); 0.384 mmole/L Ca2+, 0.096 mmole/L Mg2+, 0.148 mmole/L Na+, 0.015 mmole/L K+, 0.783 mmole/L Cl-, 0.148 mmole/L HCO3- and 0.096 mmole/L SO42-. This was then used as dilution water. The dilution water was maintained at 10° ± 1°C with a flow through thermostat and aerated to oxygen saturation.

OTHER TEST CONDITIONS
- Photoperiod: 16-hours light and 8-hours dark
- Light intensity: 256 lux

POST-HATCH DETAILS
- Begin of post-hatch period: The post-hatch period began after 90 percent of all fertilized and living eggs in the controls had hatched (study day 34, post hatch day (PHD) 0).
- No. of hatched eggs (alevins)/treatment released to the test chamber: Alevin were thinned to 15 individuals per replicate.
- Release of alevins from incubation cups to test chamber on day no.: study day 43 (post-hatch day 9).

FERTILIZATION SUCCESS STUDY
- Number of eggs used: 35 eggs
- Removal of eggs to check the embryonic development on day no.: On day 12 eggs from the fertilization control group were removed from the test and cleared in a 10% acetic acid solution for some minutes. Observations for evidence of embryonic development were made and the eggs were then discarded. Those eggs exhibiting no indication of embryonic development were deemed non-viable (non-fertilized).

Reference substance (positive control):

no

Key result

Duration:

94 d

Dose descriptor:

NOEC

Effect conc.:

83.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: based on swim up and growth effects

Remarks on result:

other:

Details on results:

The definitive study was conducted at the nominal test concentrations of 9.46 (9.40), 18.9 (18.8), 37.7 (37.5), 75.5 (75.0), 150.9 (150) and 301.8 (300) µg test substance (a.s.)/L (ppb).

The mean measured concentrations during the test were 10.2, 21.7, 40.0, 83.2, 167.4 and 345.1 µg a.s./L (ppb). These mean measured values ranged from 107 to 115 percent of nominal during the test period for all test levels. (Table 10). All reported results refer to the mean measured concentrations of Pencycuron in water.

The mean measured concentrations of all five series of stock solutions from all six test level were in good accordance with the nominal values. These mean measured values for all stock solutions concentrations ranged from 99 to 105 to percent of nominal during the test period (Table 11).

The stability of the test item in the stock solution with acetone was checked additionally. No degradation of Pencycuron was observed during a duration of 20 days (from study day - 6 to day 14) at 10°C. Therefore, Pencycuron was quite stable under conditions of testing.

Percent egg hatchability was evaluated on study day 37 (post hatch day 3). Hatching success ranged from 91 percent to 96 percent: Control (95%), Solvent Control (94%), 10.2 µg a.s./L (92%), 21.7 µg a.s./L (93%), 40.0 µg a.s./L (93%), 83.2 µg a.s./L (96%), 167.4 µg a.s./L (95%) and 345.1 µg a.s./L (91%) (Table 5). There was no significant reduction in egg hatchability in any treatment group compared to the pooled controls.

Time to hatch was evaluated for all test levels. Egg hatching began on study day 32 and continued until day 36 in all test levels and in the controls (Table 7). Between study day 34 (post hatch day 0) and 37 (post hatch day 3) there was no significant difference in hatching success in any treatment group compared to the solvent control and pooled control data, respectively.

Newly hatched fry began to swim up from the bottom of the test chambers on study day 46 (post-hatch day 12). Swim-up was observed between study day 46 and 72 (Table 8). On day 50 in the controls, more than 90% of the fry had emerged. On study day 50 (lasting up to day 53) swim-up was significantly reduced in the test levels at 167.4 µg a.s./L and 345.1 µg a.s./L compared to the pooled controls. On study day 54 (lasting up to day 67) swim-up was significantly reduced in the highest test level at 345.1 µg a.s./L.

Fry survival was analyzed on study day 94 (post-hatch day 60). On post-hatch day 60 fry survival ranged from 83 percent to 100 percent: Control (93%), Solvent Control (97%), 10.2 µg a.s./L (100%), 21.7 µg a.s./L (98%), 40.0 µg a.s./L (98%), 83.2 µg a.s./L (95%), 167.4 µg a.s./L (100%) and 345.1 µg a.s./L (83%) (Table 5). In the test level at 345.1 mg a.s./L fry survival was significantly reduced in comparison to the pooled control data.

Fry growth, expressed as length, was measured on study day 94 (post-hatch day 60; study termination). Data analysis showed statistically significant difference in comparison to the pooled control data in the test levels at 10.2 µg a.s./L, 167.4 µg a.s./L and 345.1 µg a.s./L (Table 6). The significant effect in the test level at 10.2 µg a.s./L indicates a no test item-related effect, because of the lack of a dose-response, and was excluded from the NOEC-definition.

Fry growth, expressed as dry weight, was measured on study day 94 (post-hatch day 60; study termination). Data analysis showed statistically significant difference in comparison to the pooled control data in the test levels at 167.4 µg a.s./L and 345.1 µg a.s./L (Table 6).

The egg fertilization rate was checked 12 days after fertilization with the 140 additional eggs (35 per incubation cup) which were placed in separate egg incubation cups at the test initiation. The fertilization success in the four replicates ranged from 97 to 100 percent with a mean of 99.3 percent (Table 9).

During the post hatch period between study day 58 and test termination the following morphological and behavioral effects were frequently observed in the test levels >167.4 µg a.s./L and in the other test levels only sporadically: fish laid inactive on the bottom of the aquarium, turned dark in coloration. These observations indicate test item-related effects.

See "Attachments" in "Overall remarks, attachments" for the tables.

Validity criteria fulfilled:

yes

Conclusions:

Based on the statistical analysis of egg hatch, time to hatch, time to swim-up, fry survival and growth (expressed as weight and length) the no-observed-effect-concentrations (NOEC's) and the lowest-observed-effect-concentrations (LOEC's) were determined as follows (all results listed are mean measured a.s./L concentrations): Egg hatchability at study day 37 resulted in a NOEC =345 ug a.s./L and a LOEC >345 µg a.s./L. Time to hatch from study day 34 - 37 resulted in a NOEC =345 ug a.s./L and a LOEC >345 µg a.s./L. Time to swim-up from study day 50 to 53 resulted in a NOEC at 83.2 µg a.s./L and a LOEC at 167 µg a.s./L. Fry survival on study day 94 (study termination) resulted in a NOEC at 167 µg a.s./L and a LOEC at 345 µg a.s./L. Growth at study day 94 (study termination), expressed as length, resulted in a NOEC at 83.2 µg a.s./L and a LOEC at 167 µg a.s./L. Growth at study day 94 (study termination), expressed as dry weight, resulted in a NOEC at 83.2 µg a.s./L and a LOEC at 167 µg a.s./L. Morphological and behavioural effects were frequently observed in the test levels =167.4 µg a.s./L and in other test levels only sporadically during the post hatch period which indicates test item related effects. So, the overall chronic 94-day-NOEC for Pencycuron to early life stages of rainbow trout is 83.2 µg a.s./L (based on swim-up and growth effects) and the overall 94-day-LOEC is 167 µg a.s./L (= MATC of 118 µg a.s./L).

Executive summary:

The early life stage toxicity of pencycuron to rainbow trout (Oncorhynchus mykiss) was determined. Fish were exposed to a range of nominal concentrations of 9.46 (9.40), 18.9 (18.8), 37.7 (37.5), 75.5 (75.0), 150.9 (150.0), and 301.8 (300.0) μg a.s./L (ppb), alongside a dilution water control and a solvent control.  The overall 94-day exposure to pencycuron resulted in a NOEC of 83.2 μg a.s./L (based on swim-up and growth effects).

Description of key information

A fish early life stage study was conducted to estimate the long-term toxicity of Pencycuron to rainbow trout (Oncorhynchus mykiss). Based on mean measured concentrations of Pencycuron the NOEC was determined to be 0.0832 mg/L.

 

Test species	Result	Assessment	Reference
Rainbow trout (Oncorhynchus mykiss)	94-d NOEC = 0.0832 mg a.s./L (mean measured)	Key study	Dorgerloh & Sommer (2002)
Rainbow trout (Salmo gairdneri)	21-d NOEC > 0.3 mg a.s./L	This chronic fish study performed in accordance with OECD 204 is not considered as a suitable long-term-test under REACH.	Grau (1989)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

0.083 mg/L