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

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:

13 Apr 1987 to 04 May 1987

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:

Samples were taken on day 0, 4, 7, 14 and 21 of the study.

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A stock solution of 0.262 mg/mL was prepared. To prepare the diluter stock solution, 0.060 mL of this stock solution was placed in a 100-mL volumetric flask and diluted with acetone. A portion of the diluter stock solution was placed in a graduated culture tube in association with the injector and refilled as needed. The remaining portion of the 100-mL volume of diluter stock was kept refrigerated. The concentration of the diluter stock solution was 0.157 mg/L.
The diluter system injected 0.10 mL of diluter stock solution into the mixing box (1120 mL). A 0.050 mL aliquot of acetone was injected into the solvent control which was equivalent to that injected into the highest test concentration.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Age at study initiation: < 24 h old
- Stage and instar at study initiation: first-instar (<24 hours)
- Source: in-house culture
- Feeding during test :
Food type: a suspension of algae (Selenastrum capricornutum) supplemented with a cereal leaves and yeast suspension
Amount: 20-30 mL
Frequency: 3 times a day

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

225 - 275 ppm CaCO3

Test temperature:

15 - 20 °C

pH:

7.8 - 8.3

Dissolved oxygen:

9.2 - 10.1 ppm

Conductivity:

700 µmhos/cm

Nominal and measured concentrations:

Nominal concentrations: 1.0, 1.7, 3.5, 6.0 and 14 ng/L
Mean measured concentrations: 1.6, 2.3, 4.5, 6.8 and 17 ng/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
The system contained seven sets of four replicate one-liter test chambers, designated as control, solvent control and concentration level #1 through level #5. Flow-splitting chambers were utilized to thoroughly mix and divide each concentration for delivery to the test chambers. One liter glass beakers labeled A, B, C and D, with notched drains which were covered with 50-mesh stainless steel screen to prevent escape of the daphnids, were used as the test chambers. Daphnids were placed in each of the quadruplicate chambers. Aerated, aged well water was delivered to each test chamber at a average rate of 3.9 mL/min, an amount which was sufficient to replace the 1 liter test volume approximately 5.7 times in a period. The test chambers were immersed in a temperature controlled water bath held at 20°C .
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Well water
- Intervals of water quality measurement: parameters of dissolved oxygen and pH were measured on 0, 4, 7, 14 and 21 days in the control, low, middle and high test concentrations. Temperature measurements of the water bath were made daily.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light, 8 hours dark with a 30 min transition period
- Light intensity: 50 - 70 footcandles

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Survival of the organisms was recorded daily. After first instar were observed, the reproduction success was measured by counting and discarding the offspring produced in each concentration every Monday, Wednesday and Friday for the duration of the study. Upon termination of the test on day 21 of exposure, the surviving adults were removed from the test chambers and isolated on microscope depression slides. The length of each adult daphnid was measured.

TEST CONCENTRATIONS
- Range finding study
A 10-day flow-through was run to establish an effect, no-effect range for the definitive study with the test substance. From the results of preliminary testing and discussions with the study sponsor, the test established for the chronic investigations with 14C-test substance were 1.0, 1.7, 3.5, 6.0 and 14 ppt. The test concentrations of were measured on days 0, 7, and 21 of the study through the use of liquid scintillation counting techniques.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.6 ng/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

body lenght

Duration:

21 d

Dose descriptor:

NOEC

Remarks:

Parental survival

Effect conc.:

> 17 ng/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Observations on body length: A one-way analysis of variance and Dunnett's test indicated that the daphnid lengths in the test substance mean measured concentrations of 2.3, .4.5, 6.8 and 17 ppt were significantly different from the controls
- Other biological observations: The mean young/adult/reproduction day after 21 days was significantly affected in the mean measured exposure levels of 2.3, 4.5, 6.8 and 17 ppt. Adults with ephippial eggs were observed in both the 6.8 and 17 ppt test concentrations during the study. No young were observed until 8 days into the study.
- Mortality of control: The one-way analysis of variance, used to analyze the survival, lengths and reproduction of the control and solvent control, failed to identify any significant effects. Therefore, the control data (control and solvent control) were pooled and used the analysis of all treatments.
- Mortality of test animals: After 21-day exposure to the test substance, daphnid survival in the test concentrations were not significantly different from the pooled controls

Reported statistics and error estimates:

The chronic life cycle study was a nested block design. The measured parameters of survival, surviving adult length and total young/adult/reproduction day in the quadruplicate test chambers of the control and solvent was analyzed using a one-way analysis of variance (ANOVA). If the analysis indicated no significant effect, the control and solvent control data were pooled. All treatments were compared to pooled controls (control and solvent control).
The selected parameters of adult length (pooled) and total young/adult/reproduction day were analyzed using a one-way analysis of variance. When treatment effects were indicated following a significant F-test of the mean square ratios a multiple means comparison test, Dunnett's Test was used to determine which levels differed from the control values.

Table 1. Measured Concentrations of ¹⁴C-test substance during the 21-day chronic life cycle toxicity study with Daphnia magna

	Measured Concentration (ppt)^b	Measured Concentration (ppt)^b	Measured Concentration (ppt)^b	Measured Concentration (ppt)^b	Measured Concentration (ppt)^b	Measured Concentration (ppt)^b	Measured Concentration (ppt)b
	Day 0	Day 4	Day 7	Day 14	Day 21	Mean (± S.D.)	% Nominal
Level #1 (1.0 ppt))^a	1.6	1.9	1.3	1.4	1.7	1.6 (±0.24)	160
Level #2 (1.7 ppt))^a	2.4	2.3	2.0	2.0	2.8	2.3 (±0.33)	135
Level #3 (3.5 ppt))^a	3.9	4.8	4.2	4.3	5.2	4.5 (±0.52)	129
Level #4 (6.0 ppt))^a	6.5	7.6	6.3	6.8	6.9	6.8 (±0.50)	113
Level #5 (14 ppt))^a	15	19	16	17	17	17 (±1.5)	121
Stock (0.157ug/ml)^a	0.165	0.158	0.208	0.228	0.242	0.200 (±0.0374)	127

^aNominal concentrations

^bStock concentrations in ug/ml

 

Table 2. Percent survival, adult length and young/ adult/ reproduction day of Daphnia magna continuously exposed to ¹⁴C-test substance during 21-day life cycle study

Chamber I.D. (nominal concentration)	Mean Measured Concentration (ng/L)	Mean Percent Survival	Adult Mean Length (mm)	Mean Yong/ Adult/ Reproduction Day
Control	-	98	4.0	6.1
Solvent Control	-	98	4.0	6.2
Control Pooled^a	-	98	4.0	6.2
Level #1 (1.0 ng/L))^a	1.6	100	3.9	6.2
Level #2 (1.7 ng/L))^a	2.3	100	3.8*	4.5*
Level #3 (3.5 ng/L))^a	4.5	100	3.7*	2.6*
Level #4 (6.0 ng/L))^a	6.8	100	3.5*	1.8*
Level #5 (14 ng/L))^a	17	95**	3.4*	0.90*

* Denotes values significantly different (a=0.05) from the pooled controls using one-way analysis of variance (ANOVA) and Dunnett’s Multiple means test.

** One adult was killed in transfer on day 15 and was included in compound related mortality total.

*** Represents pooled control and solvent control values. The controls were combined since statistical analysis showed no significant difference between measured parameters.

Validity criteria fulfilled:

yes

Conclusions:

In a chronic toxicity study with Daphnia magna, performed in accordanc with EPA 72-4, the NOEC for parental survival was >17 ng/L (no effects observed in all concentrations). The NOEC for reproduction and body length was 1.6 ng/L.

Executive summary:

The chronic toxicity of 14C-test substance (radiochemical purity 99%) to Daphnia magna was assessed in a 21- day flow-through study performed following the EPA 72-4 guidance and in compliance with GLP. First instar daphnids (≤24 hours old, 40 per treatment, 10 daphnia per replicate vessel) were used to initiate the study. The nominal concentrations were 1.0, 1.7, 3.5, 6.0 and 14 ng/L plus an untreated and a solvent control (acetone). Mean measured radioactivity concentrations, determined by LSC, were 1.6, 2.3, 4.5, 6.8 and 17 ng eq./L, representing 113 to 160% of nominal. Water quality parameters were in accordance with the OECD 211 guideline.

Survival of adult daphnia was not statistically significantly reduced in any of the concentrations tested. A slight delay in time to first brood (one day) was observed at 17 ng/L. Reproductive success, as measured by the mean number of live young per adult, was significantly reduced at and above 2.3 ng/L when compared to the pooled control group. Mean total body length after 21 days was significantly reduced at and above 2.3 ng/L The NOEC for parental survival was >17 ng/L. The NOEC for reproduction and body length was 1.6 ng/L.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

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:

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

Deviations:

yes

Remarks:

The test was modified to mimic environmentally realistic exposure conditions

GLP compliance:

yes

Test organisms (species):

Daphnia magna

Test type:

flow-through

Water media type:

freshwater

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

3.2 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

slight delay in time to first brood

Survival of adult daphnia of any age group was not statistically significantly reduced in any of the concentrations tested. The dry weight of surviving first and second-generation daphnids was not significantly different from the controls at any concentration. Reproductive success, as measured by the mean number of young per adult, was significantly reduced at an initial concentration of 50 µg/L (age group <24 hours old only).

A delay in time to first brood of 0.7 - 1.0 day and 1.7 - >2 days, respectively, was observed for the second generation daphnids that originated from 8-day old adults and 11-day old adults, exposed to the two highest concentrations (13 and 50 µg/L). It was reported that this difference was not statistically significant. Values for individual replicates were not reported for this parameter, contrary to all other parameters that were investigated. For all test concentrations below 13 µg/L, and for both controls, the mean first day of offspring production for the second-generation daphnids that originated from 8-day old adults and 11-day old adults was 8.0 days. Hence this parameter appeared to show little variability. The mean first day of offspring production for the second-generation daphnids that originated from 8-day old adults and 11-day old adults, respectively, was 8.7 and 9.0 days at 13 µg/L, and >10.0 and 9.7 days at 50 µg/L. A dose-relationship is therefore apparent at both test concentrations. The slight delay in the time to first brood at 13 µg/L, although reported to be not statistically significant, is considered to a treatment related effect. The NOEC from this study is 3.2 µg/L.

Validity criteria fulfilled:

yes

Conclusions:

In a chronic aquatic toxicity study in Daphnia magna, performed in accordance with EPA 72-4 with modified design, a NOEC of 3.2 µg/L was determined based on the observed slight delay in the time to first brood.

Executive summary:

The chronic toxicity of the test substance to Daphnia magna was assessed in a 21-day flow-through study performed following the EPA 72-4 guidance and in compliance with GLP. The nominal starting concentrations were 0.20, 0.80, 3.2, 13 and 50 µg/L, with untreated and solvent control (acetone). Exposure concentrations were manipulated so as to gradually achieve a reduction of 50% in concentration after each ten-hour period throughout the duration of the study. Each exposure concentration was tested in 20 daphnids (2 replicates of 10) of each of four age groups (<24 hours old, 4-6, 8 and 11 days old). Test substance concentrations were determined in centrifuged samples of test solutions of each concentration at 14 time points up to 21 days. Measured concentrations were in agreement with the expected nominal concentrations at all sampling times (except a few values near the LOQ of the analytical method): time averaged means were 86-109% of nominal, CV 6-18%. Test substance concentrations fell below the limit of detection within 3-7 days at all 5 concentration levels. Three batches of 10 offspring collected from each treatment and parental age group were transferred to dilution water without the test substance and maintained for a further 12 days to assess effects on reproduction of second-generation daphnids. Offspring were not collected during the first 4 days of exposure, when concentrations of the test substance above the LOQ may have been present, or during the first 4 days of reproduction, to avoid collecting the first brood. Assessments of survival, growth and reproduction were performed on first and second-generation daphnids. Water quality parameters were in accordance with the OECD 211 guideline.

Survival of adult daphnia of any age group was not statistically significantly reduced in any of the concentrations tested. The dry weight of surviving first and second-generation daphnids was not significantly different from the controls at any concentration. Reproductive success, as measured by the mean number of young per adult, was significantly reduced at an initial concentration of 50 µg/L (age group <24 hours old only). A delay in time to first brood of 0.7 - 1.0 day and 1.7 - >2 days, respectively, was observed for the second generation daphnids that originated from 8-day old adults and 11-day old adults, exposed to the two highest concentrations (13 and 50 µg/L). It was reported that this difference was not statistically significant. Values for individual replicates were not reported for this parameter, contrary to all other parameters that were investigated. For all test concentrations below 13 µg/L, and for both controls, the mean first day of offspring production for the second-generation daphnids that originated from 8-day old adults and 11-day old adults was 8.0 days. Hence this parameter appeared to show little variability. The mean first day of offspring production for the second-generation daphnids that originated from 8-day old adults and 11-day old adults, respectively, was 8.7 and 9.0 days at 13 µg/L, and >10.0 and 9.7 days at 50 µg/L. A dose-relationship is therefore apparent at both test concentrations. The slight delay in the time to first brood at 13 µg/L, although reported to be not statistically significant, is considered to a treatment related effect. The NOEC from this study is 3.2 µg/L.

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.

Freshwater 21-d NOEC = 1.6 ng/L, flow-through, Daphnia magna, reproduction and body length, EPA 72 -4, Forbis 1987

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

1.6 ng/L

Additional information

EPA 72 -4, Forbis 1987, Daphnia

The chronic toxicity of 14C-test substance (radiochemical purity 99%) to Daphnia magna was assessed in a 21- day flow-through study performed following the EPA 72-4 guidance and in compliance with GLP. First instar daphnids (≤24 hours old, 40 per treatment, 10 daphnia per replicate vessel) were used to initiate the study. The nominal concentrations were 1.0, 1.7, 3.5, 6.0 and 14 ng/L plus an untreated and a solvent control (acetone). Mean measured radioactivity concentrations, determined by LSC, were 1.6, 2.3, 4.5, 6.8 and 17 ng eq./L, representing 113 to 160% of nominal. Water quality parameters were in accordance with the OECD 211 guideline.

Survival of adult daphnia was not statistically significantly reduced in any of the concentrations tested. A slight delay in time to first brood (one day) was observed at 17 ng/L. Reproductive success, as measured by the mean number of live young per adult, was significantly reduced at and above 2.3 ng/L when compared to the pooled control group. Mean total body length after 21 days was significantly reduced at and above 2.3 ng/L The NOEC for parental survival was >17 ng/L. The NOEC for reproduction and body length was 1.6 ng/L.

Supporting study

EPA 72 -4, Ward 1995, Daphnia

The chronic toxicity of the test substance to Daphnia magna was assessed in a 21-day flow-through study performed following the EPA 72-4 guidance and in compliance with GLP. The nominal starting concentrations were 0.20, 0.80, 3.2, 13 and 50 µg/L, with untreated and solvent control (acetone). Exposure concentrations were manipulated so as to gradually achieve a reduction of 50% in concentration after each ten-hour period throughout the duration of the study. Each exposure concentration was tested in 20 daphnids (2 replicates of 10) of each of four age groups (<24 hours old, 4-6, 8 and 11 days old). Test substance concentrations were determined in centrifuged samples of test solutions of each concentration at 14 time points up to 21 days. Measured concentrations were in agreement with the expected nominal concentrations at all sampling times (except a few values near the LOQ of the analytical method): time averaged means were 86-109% of nominal, CV 6-18%. Test substance concentrations fell below the limit of detection within 3-7 days at all 5 concentration levels. Three batches of 10 offspring collected from each treatment and parental age group were transferred to dilution water without the test substance and maintained for a further 12 days to assess effects on reproduction of second-generation daphnids. Offspring were not collected during the first 4 days of exposure, when concentrations of the test substance above the LOQ may have been present, or during the first 4 days of reproduction, to avoid collecting the first brood. Assessments of survival, growth and reproduction were performed on first and second-generation daphnids. Water quality parameters were in accordance with the OECD 211 guideline.

Survival of adult daphnia of any age group was not statistically significantly reduced in any of the concentrations tested. The dry weight of surviving first and second-generation daphnids was not significantly different from the controls at any concentration. Reproductive success, as measured by the mean number of young per adult, was significantly reduced at an initial concentration of 50 µg/L (age group <24 hours old only). A delay in time to first brood of 0.7 - 1.0 day and 1.7 - >2 days, respectively, was observed for the second generation daphnids that originated from 8-day old adults and 11-day old adults, exposed to the two highest concentrations (13 and 50 µg/L). It was reported that this difference was not statistically significant. Values for individual replicates were not reported for this parameter, contrary to all other parameters that were investigated. For all test concentrations below 13 µg/L, and for both controls, the mean first day of offspring production for the second-generation daphnids that originated from 8-day old adults and 11-day old adults was 8.0 days. Hence this parameter appeared to show little variability. The mean first day of offspring production for the second-generation daphnids that originated from 8-day old adults and 11-day old adults, respectively, was 8.7 and 9.0 days at 13 µg/L, and >10.0 and 9.7 days at 50 µg/L. A dose-relationship is therefore apparent at both test concentrations. The slight delay in the time to first brood at 13 µg/L, although reported to be not statistically significant, is considered to a treatment related effect. The NOEC from this study is 3.2 µg/L.