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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Aug 2001 to 21 Aug 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

according to guideline

Guideline:

other: OPPTS 850.5400: Algal Toxicity, Tiers l and II

Version / remarks:

1996

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples of the test solutions were collected at approximately 0 and 96 hours to measure concentrations of the test substance. Samples at test initiation were collected from the individual batches of test solution prepared for each treatment and control group prior to distribution to the replicate test chambers. At test termination, samples were collected from the six pooled negative control replicates and three pooled replicates from each treatment. All samples were collected in glass vials. The samples were analyzed immediately without storage.

Vehicle:

yes

Remarks:

Freshwater algal medium

Details on test solutions:

A primary stock solution was prepared by dissolving the test substance in freshwater algal medium at a concentration of 40 mg/L. The stock was sonicated about 30 minutes and inverted at least 20 times to mix and appeared cloudy brown. A secondary stock was prepared by diluting 200 mL of the 40 mg/L stock solution to achieve to highest test concentration 4.0 mg/L. The secondary stock was proportionally diluted with freshwater algal medium to prepare the four additional test solutions at nominal concentrations of 0.13, 0.25, 0.50, 1.0 and 2.0 mg/L. All test solutions appeared clear and colorless.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Age of inoculum: exponential growth phase

ACCLIMATION
- Acclimation period: 14 days

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

23 ± 2°C

pH:

7.8 - 10

Nominal and measured concentrations:

The nominal concentrations were 0.13, 0.25, 0.50, 1.0, 2.0 and 4.0 mg product/L
The measured concentrations were 0.12, 0.25, 0.45, 0.92, 1.8 and 3.8 mg product/L at test initiation (representing 90-101% of nominal), and 0.04, 0.10, 0.21, 0.46, 1.0 and 3.3 mg product/L at the end of exposure (representing 32-81% of nominal), based on the product.
The test substance concentration calculation was based on a 25.6% content in the 25WG product, as reported for the same batch used for the studies in fish, daphnia and algae.
Mean measured concentrations after correction for the content: 0.01, 0.026, 0.054, 0.118, 0.265, 0.845 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile 250-mL Erlenmeyer flasks plugged with foam stoppers, and containing mL of test or control medium
- Initial cells density: 1.0 x 10^4 cells/mL
- Control end cells density: 8.25 x 10^6 cells/mL
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous
- Light intensity and quality: 6050 to 6680 lux,

EFFECT PARAMETERS MEASURED
Test medium samples were collected from each treatment group and control group for the determination of algal cell densities. Single samples from each replicate were collected at approximately 24-hour intervals during the 96-hour exposure and were held for a maximum of three days under refrigerated conditions to inhibit growth until cell counts could be performed.
Samples of test solution were collected from each of the three "biological" replicates per treatment and control group at the end of the test. These samples were pooled within their respective treatments, and subsamples were removed and examined microscopically for atypical cell morphology (e.g., changes in cell shape, size or color). Growth of cells in the replicate test chambers also was assessed for aggregations or flocculations of cells, and adherence of the cells to the test chamber.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.84 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: After correction for the content of the test substance in the formulation (3.3 mg product/L)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.38 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: After correction for the content of the test substance in the formulation (1.5 mg product/L)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: After correction for the content of the test substance in the formulation (0.45 mg product/L)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.064 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: After correction for the content of the test substance in the formulation (0.25 mg product/L)

Details on results:

- Exponential growth in the control (for algal test): yes
- Unusual cell shape: There were no noticeable changes in cell morphology at concentrations mg/L when compared to the control. However, enlarged cells were noted in the 3.8 mg product/L treatment groups at 96 hours
- Adherence to test vessels, flocculation, agregation: After 96 hours of exposure, there were no signs of adherence of cells to the test chambers or aggregation/flocculation of algae in the negative control or in any treatment group.
- Inhibition of cell density: After 72 hours of exposure, inhibition of cell density in the 0.12, 0.25, 0.45, 0.92, 1.8 and 3.8 mg product/L treatment groups was 15, 10, 28, 31, 64 and 96%, respectively, relative to the negative control. Dunnett's test showed that cell density was significantly reduced in the 0.45, 0.92, 1.8 and 3.8 mg product/L treatment groups (p<0.05). Consequently, the 72-hour NOAEC for cell density was 0.25 mg product/L. After 96 hours of exposure, inhibition of cell density in the 0.12, 0.25, 0.45, 0. 92, 1.8 and 3.8 mg product/L treatment groups was 7.5, 7.8, 16, 20, 58 and 99%, respectively relative to negative control. Dunnett's test showed that cell density was significantly reduced in the 0.45, 0.92, 1.8 and 3.8 mg product/L treatment groups (p<0.05). Consequently, the 96-hour NOAEC for cell density was 0.25 mg product/L
- Inhibition of biomass: After 72 hours of exposure, inhibition of biomass in the 0.12, 0.25, 0.45, 0.92, 1.8 and 3.8 mg product/L treatment groups was 13, 10, 28, 28, 59 and 92%, respectively, relative to the negative control. Dunnett’s test showed that biomass was significantly reduced in the 0.45, 0.92, 1.8 and 3.8 mg product/L treatment groups (p<0.05). Consequently, the 72-hour NOAEC for biomass was 0.25 mg productIL. After 96 hours of exposure, inhibition of biomass in the 0.12, 0.25, 0.45, 0.92, 1.8 and 3.8 mg product/L treatment groups was 10, 8.9, 21, 24, 59 and 97%, respectively, relative to the negative control. Dunnett’s test showed that biomass was significantly reduced in the 0.45, 0.92, 1.8 and 3.8 mg product/L treatment groups (p<0.05). Consequently, the 96-hour NOAEC for biomass was 0.25 mg product/L
- Inhibition of growth rate: After 72 hours of exposure, inhibition of growth rate in the 0.12, 0.25, 0.45, 0.92, 1.8 and 3.8 mg product/L treatment groups was 2.7, 2.1, 5.8, 6.5, 18 and 60%, respectively, relative to the negative control. Dunnett’s test showed that growth rates were significantly reduced in the 0.92, 1.8 and 3.8 mg product/L treatment groups (p<0.05). Consequently, the 72-hour NOAEC for growth rate was 0.45 mg productIL. After 96 hours of exposure, percent inhibition of growth rate in the 0.12, 0.25, 0.45, 0.92, 1.8 and 3.8 mg product/L treatment groups was 1.1, 1.3, 2.6, 3.3, 13 and 67%, respectively, relative to the negative control. Dunnett’s test showed that growth rates were significantly reduced in the 1.8 and 3.8 mg product/L treatment groups (p<0.05). Consequently, the 96-hour NOAEC for growth rate was 0.92 mg product/L.

Reported statistics and error estimates:

The data were evaluated for normality and homogeneity of variance (p=0.05) using Shapiro-Wilk’s and Levene's tests, respectively. If the assumptions of normality and homogeneity of variances were not met an attempt was made to correct the condition by log transformation of the data. In some instances, the data transformation did not correct the problem. In these cases, Dunnett's test still was used to make the comparison. The treatment groups then were compared to the control using Dunnett’s test (p=0.05).

Validity criteria fulfilled:

yes

Conclusions:

In an aquatic toxicity study in algae, performed in accordance with OECD 201, for biomass the 72h-EbC50 was 0.38 mg/L (1.5 mg product/L), and for growth the 72h-ErC50 was 0.84 mg/L (3.3 mg product/L). The 72h-NOEbC was reported to be 0.064 mg/L (0.25 mg product/L) and the 72h-NOErC was 0.12 mg/L (0.45 mg product/L).

Executive summary:

The objective of this study was to evaluate the toxicity of the test substance to the freshwater green alga, Pseudokirchneriella subcapitata, over a 96-hour exposure period under static test conditions in a test performed following the OECD TG 201 and in compliance with GLP. The freshwater green alga, Pseudokirchneriella subcapitata, was exposed to a geometric series of six test concentrations and a negative (culture medium) control under static conditions for 96 hours. Six replicates in the negative control and three replicates per treatment was tested. Nominal test concentrations were 0.13, 0.25, 0.50, 1.0, 2.0 and 4.0 mg product/L. Mean measured concentrations were determined from samples of test medium collected from each treatment and control group at the beginning and end of the test. At test initiation an inoculum of algal cells was added to each test chamber to achieve a nominal concentration of approximately 10,000 cells/ml. Samples were collected from each test chamber at approximately 24-hour intervals during the test to determine cell densities, which were subsequently used to calculate areas under the growth curve and growth rates. Cell densities, areas under the growth curve (biomass) and growth rates were used to calculate percent inhibition values relative to the control over the 96-hour exposure period.  ECb50, ECr50 and EC50 values were calculated, when possible, based upon cell density, biomass and growth rate, respectively, for each 24-hour interval of the exposure No observed adverse effect concentrations (NOAEC) were determined at 72 and 96 hours through statistical evaluation of the cell densities, biomass and growth rates, as well as examination of the concentration-response pattern. The results were: 72h-EbC50: 1.5 mg product/L (after correction for the content of the test substance in the formulation: 0.38 mg/L), 72h-ErC50: 3.3 mg product/L (after correction for the content of the test substance in the formulation: 0.84 mg/L), 72h-NOEbC: 0.25 mg product/L (after correction for the content of the test substance in the formulation: 0.064 mg/L) and 72h-NOErC: 0.45 mg product/L (after correction for the content of the test substance in the formulation: 0.12 mg/L).

Reference 2

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Test type:

static

Water media type:

freshwater

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

1.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

< 0.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

The measured concentrations were 1.2, 1.6 and 2.0 mg/L at test initiation (representing 67-120% of nominal), and 0.2, 1.2 and 0.9 mg/L at the end of exposure (representing 20-75% of nominal) for the nominal concentrations of 1.0, 1.6 and 3.0 mg/L, respectively. Endpoints were based on nominal concentrations, which is not acceptable. Water quality parameters were in accordance with the OECD guideline 201.

Table 1. The acute toxicity of the test substance to algae

Species	Test type and duration. (purity of test substance)	Actual concn. (as % of nominal)	LC/EC50 in mg/L	NOEC mg/L
Scenedesmus subspicatus (green alga)	Static 96 hours (96.6%)	20 - 120	96h-EbC50: 1.1(A)	96h-NOEC: <0.8(A)

(A) Based on nominal concentrations

Validity criteria fulfilled:

no

Conclusions:

In an acute aquativ toxicity test performed in accordance with OECD TG 201, the 96-hour EbC50 was determined to be 1.1 mg/L, and the 96-hour NOEC: <0.8 mg/L; all based on nominal concentrations.

Executive summary:

A 96-hour toxicity test on green algae (Scenedesmus subspicatus) was performed according to OECD TG 201 (with restrictions) in 3 flasks per concentration each initially containing 1.1 x 10^4 cells/mL. The test was conducted with the test substance at nominal test concentrations of 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0 and 3.0 mg/L, and an untreated control. The measured concentrations were 1.2, 1.6 and 2.0 mg/L at test initiation (representing 67 -120% of nominal), and 0.2, 1.2 and 0.9 mg/L at the end of exposure (representing 20-75% of nominal) for the nominal concentrations of 1.0, 1.6 and 3.0 mg/L, respectively. Endpoints were based on nominal concentrations.

The 96 -hour EbC50 was determined to be 1.1 mg/L, and the 96-hour NOEC: <0.8 mg/L; all based on nominal concentrations.

Description of key information

All available data was assessed and the studies representing the worst-case effects are included here as key. The results can be considered worst-case and are selected for the CSA. Other studies are included as supporting information.

Freshwater, 96-h NOEbC = 0.064 mg/L, static, Pseudokirchneriella subcapitata, OECD TG 201, Desjardins 2001

Freshwater, 96-h EbC50 = 0.38 mg/L, static, Pseudokirchneriella subcapitata,OECD TG 201, Desjardins 2001

Key value for chemical safety assessment

EC50 for freshwater algae:

0.38 mg/L

EC10 or NOEC for freshwater algae:

0.064 mg/L

Additional information

OECD TG 201, Desjardins 2001

The objective of this study was to evaluate the toxicity of the test material (Fenoxycarb 25WG) to the freshwater green alga, Pseudokirchneriella subcapitata, over a 96-hour exposure period under static test conditions in a test performed following the OECD TG 201 and in compliance with GLP. The freshwater green alga, Pseudokirchneriella subcapitata, was exposed to a geometric series of six test concentrations and a negative (culture medium) control under static conditions for 96 hours. Six replicates in the negative control and three replicates per treatment was tested. Nominal test concentrations were 0.13, 0.25, 0.50, 1.0, 2.0 and 4.0 mg product/L. Mean measured concentrations were determined from samples of test medium collected from each treatment and control group at the beginning and end of the test. The concentrations ranged from 90 – 101% of nominal at the beginning of the test to 32 to 81% of nominal at test termination. The results are based on the initial (day 0) measured concentrations 0.12, 0.25, 0.45, 0.92, 1.8 and 3.8 mg/L. At test initiation an inoculum of algal cells was added to each test chamber to achieve a nominal concentration of approximately 10,000 cells/ml. Samples were collected from each test chamber at approximately 24-hour intervals during the test to determine cell densities, which were subsequently used to calculate areas under the growth curve and growth rates. Cell densities, areas under the growth curve (biomass) and growth rates were used to calculate percent inhibition values relative to the control over the 96-hour exposure period.  EC50 values were calculated, when possible, based upon cell density, biomass and growth rate, respectively, for each 24-hour interval of the exposure No observed adverse effect concentrations (NOAEC) were determined at 72 and 96 hours through statistical evaluation of the cell densities, biomass and growth rates, as well as examination of the concentration-response pattern. The results were: 72h-EbC50: 1.5 mg product/L (after correction for the content of the test substance in the formulation: 0.38 mg/L), 72h-ErC50: 3.3 mg product/L (after correction for the content of the test substance in the formulation: 0.84 mg/L), 72h-NOEbC: 0.25 mg product/L (after correction for the content of the test substance in the formulation: 0.064 mg/L) and 72h-NOErC: 0.45 mg product/L (after correction for the content of the test substance in the formulation: 0.12 mg/L).

Supporting study

OECD TG 201, Ellgehausen 1984

A 96-hour toxicity test on green algae (Scenedesmus subspicatus) was performed according to OECD TG 201 (with restrictions) in 3 flasks per concentration each initially containing 1.1 x 10^4 cells/mL. The test was conducted with the test substance at nominal test concentrations of 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0 and 3.0 mg/L, and an untreated control. The test concentrations were prepared from stock solutions in acetone with a final acetone concentration of 0.01% in the test solutions. The measured concentrations were 1.2, 1.6 and 2.0 mg/L at test initiation (representing 67 -120% of nominal), and 0.2, 1.2 and 0.9 mg/L at the end of exposure (representing 20-75% of nominal) for the nominal concentrations of 1.0, 1.6 and 3.0 mg/L, respectively. Endpoints were based on nominal concentrations. The 96-hour EbC50 was determined to be 1.1 mg/L, and the 96-hour NOEC: <0.8 mg/L; all based on nominal concentrations.