Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

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:

24 Aug 1989 to 28 Nov 1989

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)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

The measured concentrations of the test substance in aquatic test water were determined on study days 0, 1, 7, 14 and weekly thereafter up to and day 96, which was study termination day. Control and test substance fortified water samples were also determined on each sample day. Concentrations of the test substance on these days were measured through the use of high performance liquid chromatography. At each sample day, samples were acquired by collecting approximately 25 milliliters from both replicates A and B or C and D of each test concentration using a beaker and graduated cylinder, and composited to obtain a total of 50 milliliters (A and B replicates were sampled on days 0, 7, 21, 35, 49, 63, 77 and 90; C and D replicates were sampled on days 1, 14, 28, 42, 56, 70, 84 and 96).

Vehicle:

yes

Remarks:

Dimethylformamide (DMF)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The diluter stock solution was prepared by diluting 0.818 g of test material to a 0.050 liter volume with dimethylformamide (DMF). A volume of DMF equivalent to the concentration in Level 5 was delivered to the solvent control (0.0069 mL/L).
At a purity of 94.75 %, the 0.818 g contained 0.775 g of the test substance (0.94757 X 0.818 g = 0.775 g = 775 mg). The 775 mg of the test substance dissolved in 0.05 liter of DMF yielded at 15,500 mg/L solution. The reservoir of stock on the test system was stored in an amber bottle and was connected to the syringe injector via teflon tubing. Approximately 25 mL of stock was added to the test system at a time. The remainder was stored in a refrigerator and was later added when the level in the amber bottle became low. Generally, 50 to 60 mL of stock was used weekly. Nominal exposure levels utilizing the 50% proportional diluter system based on the Level 5 concentration of 0.10 mg/L were 0.10, 0.05, 0.025, 0.013 and 0.0063 mg/L.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
Without allowing contact with water, the eggs were gently poured into a dry plastic pan and the milt was thoroughly mixed the eggs. After addition of the milt, enough 8°C control water was added to cover the eggs and the mixture was gently stirred to insure fertilization. Approximately 60 seconds after mixing, the eggs were rinsed with control water several times then covered again with water and allowed to water harden for ~2 hours. While water hardening the eggs were acclimating to test temperature (10 ± 1.5 °C) They were then distributed to the test system incubation cups.

POST-HATCH FEEDING
- Start date: Day 53
Feeding began on day 53, 17 days post-hatch. Initially, the fry were fed live brine shrimp nauplii. Ground salmon starter was added to their diet on day 56. The fish were generally fed 3 times per day. The food used during the study included Salmon Starter and brine shrimp nauplii.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 d

Hardness:

36 - 240 mg/L CaCO3

Test temperature:

9.1 - 11.8°C

pH:

7.7 - 8.2

Dissolved oxygen:

9.0 - 10.5 mg/L

Conductivity:

80 - 400 µMhos/cm

Nominal and measured concentrations:

Nominal concentrations: 0.0063, 0.013, 0.025, 0.05 and 0.10 mg/L
Mean measured concentrations: 0.0059, 0.012, 0.022, 0.048 and 0.092 mg/L

Details on test conditions:

TEST SYSTEM
- Embryo cups: incubator cups, 9.0 cm diameter X 14 cm high glass tubing with screening (16 mesh) silicone glued to the bottom. To insure exchange of water, the egg cups were oscillated vertically (3 to 6 cm) in the test solution and/or water by means of a rocker arm apparatus driven by a low rpm electric motor.
- Test vessel: exposure aquaria, approximately 15.6 X 30.7 cm with a water depth of approximately 25 cm, yielding an approximate 12 liter replicate chamber volume. Each replicate test aquarium drain was covered with 16 mesh stainless steel screen to prevent escape of the rainbow trout fry. For the first 77 days of the 96 day study, water/test solution was delivered to the 12 liter replicate chambers at an average rate of approximately 78.5 L/replicate/day. The flow rate was increased during the last 19 days to approximately 136 L/replicate/ day as a precaution against the increased oxygen demand that larger fry place on the test water. The aquaria were immersed in a water bath held at ~10°C
- Type of flow-through: proportional diluter
- No. of fertilized eggs per vessel: 35
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4 (50 eggs)
- No. of vessels per vehicle control (replicates): 4 (50 eggs)

TEST MEDIUM / WATER PARAMETERS
Water quality parameters of dissolved oxygen, temperature, conductivity, pH, hardness and alkalinity were measured in the control and treated aquaria on a weekly basis. Temperature was also measured twice daily from a single test chamber with a mercury thermometer as well as being continuously monitored with a data logger. Dissolved oxygen and temperature were measured in A replicate chambers on the designated sample days.

OTHER TEST CONDITIONS
- Photoperiod: All aquaria were illuminated by incandescent and wide spectrum fluorescent bulbs during a 16-hour daylight photoperiod, after the embryos had hatched into fry.
- Light intensity: 120 ± 17.8 footcandles

EFFECT PARAMETERS MEASURED:
Egg mortality, as discerned by a distinct change in coloration, was recorded daily and dead eggs were removed to prevent fungal- growth.
After 12-days of exposure, the eggs reserved for viability (fertilization success) determination were removed and placed in a 10% glacial acetic acid solution. After several minutes in the solution the embryos became clear. Fertilization and embryo development were indicated by the presence of a neural keel, which was visible as a white line. The percent viability was determined by dividing the number of embryos with neural keels by 50 and then multiplying by 100. The mean percent viability for the 4 replicates was then calculated. When hatching commenced, the number of embryos hatched in each incubation cup was recorded daily until day 39. Hatch was determined to be 2.95% complete on day 36 in the control.
For this reason, study day 36 became day O for the 60 day post-hatch growth period. The number of larval fry was reduced to 15 per replicate on day 39 (day 3 post-hatch). The fry were released from the incubation cups into the growth chambers on day 48 (12 days post-hatch). The fry were monitored for abnormal (sublethal) behavioral or physical changes and mortality by visually inspecting each growth chamber daily and recording the data. Survival data were collected for statistical analysis on both growth measurement days (days 35 and 60 post-hatch).
At test termination, study day 96 (60 days post-hatch) all surviving fish were sacrificed in tricane methanesulfonate. They were then blotted on paper towels to remove excess moisture and weighed. The weights were entered on a computer worksheet via direct data capture. Lengths were then measured again.


VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
Based on the results of a previous definitive acute flow-through toxicity test, 0.10 mg/L was chosen as the high test concentration. Therefore the five nominal test concentrations for the post-hatch rainbow trout early life stage investigation· were: 0.0063, 0.013, ·0.025, 0.050 and 0.10 mg/L, which correspond to test levels 1, 2, 3, 4 and 5, respectively

Reference substance (positive control):

no

Key result

Duration:

96 d

Dose descriptor:

NOEC

Effect conc.:

0.048 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

body lenght

Details on results:

- Study Initiation/Viability: Newly fertilized rainbow trout eggs (fertilized <8 hours before test initiation) were used for the initiation of this study. After 12-days the viability of the additional 200 eggs (50 per replicate) placed in the control chambers at initiation was determined by clearing them with acetic acid. The control chambers contained only dilution water during this study. The formation of a neural keel appearing as a thin white line in the cleared eggs indicated the success of fertilization. Viability of these additional eggs from 96 to in the replicates. The mean viability was 99 %. It should be noted that the egg fertilization procedure took place in control dilution water outside of the test system just prior to study and that the additional eggs for the viability test were incubated in control dilution water. Therefore, the test material no influence on the results of the viability test.
- Time to Hatch: Hatch began on day 32 and continued until day 39. On day 39, seven eggs remained in the test system, scattered through the control and four of the test levels. In one of the eggs partial embryo development was observed, but in the remaining six no development was seen. These 7 unhatched eggs were removed from the test system on day 39. No effect in the time to hatch was noted in the exposure concentrations compared to control.
- Swim-up: Newly hatched fry began swimming up from the bottom of the test chambers at 13 days post-hatch (day 49). The number of fry swimming up in each chamber was recorded for days 49 - 55. There were no obvious differences in time to swim-up in any of the test chambers. Also, no sublethal physical or behavioral effects were noted at any time during the study in any of the test concentrations.
- Hatchability: As discussed above, egg viability was determined from a viability test that indicated mean viability to be 99%. Therefore, hatch calculations were based upon 35 viable eggs (35 X 0.99 = 35) per replicate for 140 per test level (4 X 35 = 140) at initiation.
- Fry survival: Analysis of the 35 and 60 day post-hatch data indicated that fry survival in the exposure aquaria was not significantly reduced (P<0.05) when compared to the control.
- Effects on Length and Weight -- Fry growth was analyzed at 2 points during the study: length on day 35 post-hatch and length and weight on day 60 post-hatch. A growth reduction was indicated only in the Level-5 fish in the day 60 post-hatch analysis by Tukey's and Dunnett's tests. No growth effect was indicated in the day 35 post-hatch analysis. Bartlett's test indicated homogeneity of group variance for both the day 35 and day 60 post-hatch growth data. The significant effects that occurred in the exposure concentrations when compared to the control were reduced fry length and weight in the 0.092 mg/ concentrations by the end of the 60 day post-hatch period.

Reported statistics and error estimates:

Dichotomous data were analyzed by 2 X. 2 contingency tables pairing the control responses to each exposure level. An analysis of variance (ANOVA) was performed to determine if a significant difference (P<0.05) existed between groups. The data were then analyzed using Dunnett 's mean comparison test which compared the control to the test levels. Continuous data were assessed by analysis of variance techniques for nested design experiments in a manner similar to that described by McClave et al. Statistically significant differences (P<0.05) in the continuous data were determined by the calculations; Tukey's HSD and Dunnett's mean comparison.tests were used to determine those treatment levels having responses significantly different from the control response. Length and weight data were entered as replicate means for the and Dunnett's test, which were performed using the Toxstat program. Individual fish lengths and weights were the basis for the ANOVA and Tukey's test.

Table 1. Measured Concentrations of the test substance During the 96 Day Early Life Stage Toxicity Study with Rainbow Trout (Oncorhynchus mykiss)

Day	Control^a	Solvent Control^a	Level 1 (0.0063 mg/L)^b	Level 2 (0.013 mg/L)^b	Level 3 (0.025 mg/L)^b	Level 4 (0.050 mg/L)^b	Level 5 (0.10 mg/L)^b	Stock (15.550 mg/L)^b
0	<0.0026	<0.0026	0.0059	0.010	0.020	0.043	0.084	16900
1	<0.0026	<0.0026	0.0049	0.014	0.021	0.057	0.095	14800
7	<0.0026	<0.0026	0.0051	0.011	0.021	0.048	0.098	14000
14	<0.0029	<0.0029	0.0057	0.026	0.023	0.076	0.094	16200
21	<0.0029	<0.0029	0.0056	0.012	0.022	0.046	0.087	14500
28	<0.0029	<0.0029	0.0056	0.012	0.022	0.046	0.098	15500
35	<0.0029	<0.0029	0.0077	0.015	0.029	0.048	0.089	10100
42	<0.0029	<0.0029	0.0038	0.0089	0.019	0.038	0.096	15200
49	<0.0029	<0.0029	0.0069	0.011	0.023	0.048	0.098	14400
56	<0.0029	<0.0029	0.0055	0.0074	0.018	0.039	0.087	14500
63	<0.0029	<0.0029	0.0060	0.011	0.023	0.045	0.092	15300
70	<0.0029	<0.0029	0.0057	0.010	0.022	0.044	0.093	14400
77	<0.0029	<0.0029	0.0056	0.014	0.022	0.048	0.087	15600
84	<0.0029	<0.0029	0.0081	0.011	0.027	0.046	0.091	13800
90	<0.0029	<0.0029	0.0056	0.010	0.022	0.045	0.088	15300
96	<0.0029	<0.0029	0.0068	0.010	0.023	0.056	0.095	15300
Mean			0.0059	0.012	0.022	0.048	0.092	14700
Standard Deviation			±0.001	±0.004	±0.003	±0.009	±0.005	±1470
Coefficient of Variation			16.9%	33.3%	13.6%	18.8%	5.4%	10%
Mean % of Nominal			94	92	88	96	92	95

Mean ± S.D. for Levels 1-5 = 92.8 ± 2.86%

^aLess than values indicate no peak observed in control water and expressed as being below the level of sensitivity for that analysis day.

^bNominal concentration.

 

Table 2. Egg Hatchability, Fry Survival, Standard Length and Wet Weight of Rainbow Trout (Oncorhynchus mykiss)

Mean Measured Concentrations (mg/L)	35 days post-hatch	35 days post-hatch	35 days post-hatch	60 days post-hatch	60 days post-hatch	60 days post-hatch
	Hatchability^a(% hatch)	Fry survival (%)	Mean Stan. Length^b& Stan. Deviation (mm)	Fry survival (%)	Mean Stan. Length^b& Stan. Deviation (mm)	Mean Wet Weight & Stan. Deviation (g)
Control	97	100	29.8±1.71	100	45.2 ±2.62	1.38 ±0.246
Solvent Control	99	100	30.2 ±2.02	100	45.0 ±3.11	1.40 ±0.301
0.0059	96	100	29.4 ±1.48	100	43.7 ±3.53	1.31 ±0.291
0.012	96	100	29.9 ±1.86	100	44.7 ±3.47	1.35 ±0.315
0.022	96	97	29.7 ±1.95	97	43.9 ±3.08	1.27 ±0.284
0.048	96	100	30.0 ±1.67	100	44.2 ±2.76	1.27 ±0.240
0.092	99	100	29.7 ±1.64	97	43.2 ±2.58*	1.18 ±0.242*

^aPercents taken from data gathered on study day 39 when fry were thinned to 15 per replicate.

^bLength measurements taken using slides projected on a digitizing tablet and measured electronically.

* Represents significant statistical reduction (P<0.05) when compared to control.

Validity criteria fulfilled:

yes

Conclusions:

In an early life stage study with rainbow trout, performed in accordance with EPA 72-4, the significant effects that occurred in the exposure concentrations when compared to the control were reduced fry length and weight in the 0.092 mg/L concentrations by the end the 60 day post-hatch growth phase. Egg hatchability was not significantly reduced in any exposure. Survival was also not significantly reduced in any exposure level. Therefore, based on the data for this 60-day post-hatch rainbow trout early life stage toxicity study the no observed effect concentration (NOEC) was 0.048 mg/L and 0.092 mg/L was the lowest observed effect concentration (LOEC).

Executive summary:

A flow-through early life stage study with rainbow trout, Oncorhynchus mykiss, exposed to the test substance was conducted following the EPA 72-4 guidance and in compliance with GLP. Newly fertilized eggs (fertilized <8 hours before study initiation) were used for the initiation of the study with exposure continuing for 60-days post-hatch. A two-liter proportional diluter system was used to maintain constant test concentrations. Exposure concentrations of the test substance were determined through the use of HPLC. The mean measured concentrations of the test substance were: 0.0059, 0.012, 0.022, 0.048 and 0.092 mg/L. These values ranged from 88% to 96% of the nominal test concentrations of 0.0063, 0.013, 0.025, 0.050 and 0.10 mg/L. Hatchability, fry survival and growth were statistically analyzed. Comparison was made between control and test concentrations to determine if significant reductions had occurred for these parameters. No significant reduction occurred for the parameter of egg hatchability. Viability of the eggs used to initiate this study averaged 99%. Fry survival at both 35 and 60 days post-hatch was not significantly reduced at any test concentration. Rainbow trout fry growth was determined on day 35 post-hatch as a function of standard length and on day 60 post-hatch as a function of both standard length and wet weight. No growth effect was indicated in any test concentration for the day 35 post-hatch analysis. An effect of both length and weight were indicated for the day 60 post-hatch analysis in the 0.092 mg/L test concentration. No sublethal effects (abnormal behaviour or appearance) were recorded during the fry growth stage.

Based on these the no observed effect concentration (NOEC) was 0.048 mg/L and 0.092 mg/L was the lowest observed concentration (LOEC).

Description of key information

All available data was assessed and the study representing the worst-case effect is included here as key. The result can be considered worst-case and is selected for the CSA.

60-d NOEC = 0.048 mg/L, 60 -d LOEC = 0.092 mg/L, flow-through, Oncorhynchus mykiss, larval growth, EPA 72 -4, Thompson 1990

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

0.048 mg/L

Additional information

EPA 72 -4, Thompson 1990, rainbow trout

A flow-through early life stage study with rainbow trout, Oncorhynchus mykiss, exposed to the test substance was conducted following the EPA 72-4 guidance and in compliance with GLP. Newly fertilized eggs (fertilized <8 hours before study initiation) were used for the initiation of the study with exposure continuing for 60-days post-hatch. A two-liter proportional diluter system was used to maintain constant test concentrations. Exposure concentrations of the test substance were determined through the use of HPLC. The mean measured concentrations of the test substance were: 0.0059, 0.012, 0.022, 0.048 and 0.092 mg/L. These values ranged from 88% to 96% of the nominal test concentrations of 0.0063, 0.013, 0.025, 0.050 and 0.10 mg/L. Hatchability, fry survival and growth were statistically analyzed. Comparison was made between control and test concentrations to determine if significant reductions had occurred for these parameters. No significant reduction occurred for the parameter of egg hatchability. Viability of the eggs used to initiate this study averaged 99%. Fry survival at both 35 and 60 days post-hatch was not significantly reduced at any test concentration. Rainbow trout fry growth was determined on day 35 post-hatch as a function of standard length and on day 60 post-hatch as a function of both standard length and wet weight. No growth effect was indicated in any test concentration for the day 35 post-hatch analysis. An effect of both length and weight were indicated for the day 60 post-hatch analysis in the 0.092 mg/L test concentration. No sublethal effects (abnormal behaviour or appearance) were recorded during the fry growth stage.

Based on these data the no observed effect concentration (NOEC) was 0.048 mg/L and 0.092 mg/L was the lowest observed concentration (LOEC).