1,6-hexanediyl-bis(2-(2-(1-ethylpentyl)-3-oxazolidinyl)ethyl)carbamate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2003-04-01 to 2003-04-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Directive 92/69/EEC (O.J. No. L383A, 29.12.92)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Sampling schedule of chemical analysis for control: at 0 and 72 h
- Sampling schedule of chemical analysis for test concentrations: at 0 and 72 h

Vehicle:

no

Details on test solutions:

Pre-treatment of the test item:
To give the desired series of test concentrations direct weightings were prepared for the test concentrations 7.7, 15.8, 31.4, 62.1 and 125.2 mg/L. The test item was added to dilution water and treated with an ultra turrax for 60 seconds and 8000 rpm and afterwards stirred for 24 hours on a magnetic stirrer. Finally, undissolved particles of the test item were removed by filtration using folded filters of pore size 7 - 12 µm.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desomodesmus subspicatus
- Source: Non-axenic strain of the test species obtained from "The Collection of Algal Cultures" of the Institute of Plant Physiology at the University of Göttingen (Germany)

Maintenance of stock cultures: Exponentially-growing stock cultures are maintained in the test facility under the following conditions:
- constant temperature conditions (23 ± 2 °C)
- light intensity: 60 - 120 µE x m-2 x s-1 (measured in the range 400 - 70 nm using a spherical quantum flux meter)
- nutrient medium (according to Bringmann and Kühn, 1977) was renewed one a week
- Cell density measurements were made using a microcell counter

Preparation of pre-cultures:
Pre-cultures were set up three days before the start of a test. They are grown under identical exposure conditions as the stock cultures, except of the use of a different nutrient medium.

Test cultures:
The algal inocula for a test is taken form an exponentially-growing pre-culture and are mixed with the nutrient medium to make up to a final cell density of about 1E+04 cells per millilitre in the test medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 - 25 °C

pH:

The pH was measured at the beginning of the test and at 72 h.

Nominal and measured concentrations:

Nominal concentrations: 6.25, 12.5, 25, 50 and 100 mg/L. No measured test item concentrations. Only DOC measurements are available.

Details on test conditions:

Exposure conditions:
- Test vessel: 300 mL Erlenmeyer flasks with stoppers
- Culturing apparatus: Light chamber with a temperature in the range 21 - 25 °C (maintenance: ± 2 °C) and continuous uniform illumination provided in the spectral range 400 - 700 nm.
- Light intensity: At the average of the test solutions, a light intensity in the range 60 - 120 µE/m2s, or an equivalent range of 4000 - 8000 lx was recommended to use.
- Cell density measurements: Cell densities are measured in a microcell counter or, alternatively, were determined by means of a microscopic counting chamber.
- Experimental design: 5 test concentrations plus 1 control, 3 replicates per concentrations, 6 replicate per control, initial cell density in the test cultures approx. 1E+04 cells per millilitre, additionally highest test concentration without algae
- Method of administration: direct weighing

The cell density in the control cultures should increase by a factor of at least 16 within 72 h.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

43 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Analysis of the growth and growth rate of the algal population within the 72 h exposure period were done using probit analysis and Dunnett's test, respectively.
The results are expressed as effective loadings. Considering the heterogeneous composition of the test item, Water Accommodated Fractions (WAFs) were tested. The results of DOC determinations reflect rough impression only of exposure conditions over time. No information is available on a molecular weight of the test item and a potential correlation between effective loadings and measured DOC values.


Mean cell density during the test:
Nominal concentration (mg/L) / cell density (cells/mL) after 24 h, 48 h, 72 h:
- Control / 43333, 162778, 345000
- 6.25 / 54444, 214444, 387778
- 12.5 / 54444, 212222, 380000
- 25 /46667, 108889, 152222
- 50 / 30000, 40000, 44444
- 100 / 14444, 16667, 20000

Mean growth (b) (integral of biomass):
Nominal concentration (mg/L) / area under growth curve / inhibition (+); increase (-) (%)
- Control / 353611 / 0.0
- 6.25 / 437778 / -23.8
- 12.5 / 431667 / -22.1
- 25 / 206667 / 41.6
- 50 / 67222 / 81.0
- 100 / 16111 / 95.4

Mean growth rate (r):
Nominal concentration (mg/L) / growth rate (1/d) / inhibition (+); increase (-) (%)
- Control / 1.18 / 0.0
- 6.25 / 1.22 / -3.3
- 12.5 / 1.21 / -2.7
- 25 / 0.91 / 23.1
- 50 / 0.50/ 57.9
- 100 / 0.23 / 80.4

Determinations of NOEC and LOEC based on growth (b) and growth rate (r) according to:

- Dunnett, C.W. (1955): A multiple comparison procedure for comparing several treatments with a control.-Amer. statist. Ass.J. 50:1096 -1121.

- Dunnett, C.W. (1964): New tables for multiple comparisons with a control.- Biometrics 20: 482 -491.

Validity criteria fulfilled:

yes

Conclusions:

In this 72-hour toxicity test with freshwater algal species Desmodesmus subspicatus, the EbC50 and ErC50 values were determined to be 29 mg/L and 43 mg/L (nominal concentrations, WAF), respectively. The no-observed-effect concentration (NOEC) and lowest-observed-effect-concentration was determined to be 12.5 mg/L and 25 mg/L (both nominal concentrations, WAF), respectively.

Executive summary:

A study was performed to assess adverse effects of the test item on the growth (= increase in cell density) and the growth rate (=rate of increase of cell density with time) of the planktonic freshwater algal species Desmodesmus subspicatus over several generations. The study was conducted in accordance with EEC methods for Determination of Ecotoxicity Annex to directive 92/69/EEC Part c, Method 3 which is in most parts equivalent to the OECD guideline for testing of chemicals no. 201.

Exponentially growing algal cells were exposed for a period of 72 h to a range of concentrations, nominally 6.25, 12.5, 25, 50 and 100 mg/L of the test item dissolved in water. Auxiliaries used to prepare the test media were an ultra turrax, a magnetic stirrer and folded filters. The cell densities were measured at 24 h intervals. Inhibition of the algal population was measured as reduction in growth (index b) and growth rate (index r) relative to control cultures grown under identical conditions. Growth and growth rates were used to calculate a No Observed Effect Concentration and a Lowest Observed Effect Concentration according to Dunnett (1994, 1995). The following values were determined: EbC50 (72 h)=29 mg/L; ErC50 (72 h)=43 mg/L; NOEC (b)=12.5mg/L; LOEC (b)= 25 mg/L; NOEC (r)= 12.5 mg/L and LOEC (r)=25 mg/L. The results were expressed in terms of effective loadings. Considering the heterogeneous composition of the test item, Water Accommodated Fractions (WAF) were tested. The results of DOC determinations reflect a rough impression only of exposure conditions over time. No information is available on a molecular weight of the test item and on a potential correlation between effective loadings and measured DOC values.

Description of key information

The toxicity to algae was assessed according to EU method C.3. In a 72-hour toxicity test with freshwater algal species Desmodesmus subspicatus, the EbC50 and ErC50 values were determined to be 29 mg/L and 43 mg/L (nominal concentrations, WAF), respectively. The no-observed-effect concentration (NOEC) and the lowest-oberved-effect-concentration (LOEC) was determined to be 12.5 mg/L and 25 mg/L (both nominal concentrations, WAF), respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

43 mg/L

EC10 or NOEC for freshwater algae:

12.5 mg/L

Additional information

A study was performed to assess adverse effects of the test item on the growth (= increase in cell density) and the growth rate (=rate of increase of cell density with time) of the planktonic freshwater algal species Desmodesmus subspicatus over several generations. The study was conducted in accordance with EEC methods for Determination of Ecotoxicity Annex to directive 92/69/EEC Part c, Method 3 which is in most parts equivalent to the OECD guideline for testing of chemicals no. 201.

Exponentially growing algal cells were exposed for a period of 72 h to a range of concentrations, nominally 6.25, 12.5, 25, 50 and 100 mg/L of the test item dissolved in water. Auxiliaries used to prepare the test media were an ultra turrax, a magnetic stirrer and folded filters. The cell densities were measured at 24 h intervals. Inhibition of the algal population was measured as reduction in growth (index b) and growth rate (index r) relative to control cultures grown under identical conditions. Growth and growth rates were used to calculate a No Observed Effect Concentration and a Lowest Observed Effect Concentration according to Dunnett (1994, 1995). The following values were determined: EbC50 (72 h) = 29 mg/L; ErC50 (72 h) = 43 mg/L; NOEC (b) = 12.5mg/L; LOEC (b) = 25 mg/L; NOEC (r) = 12.5 mg/L and LOEC (r) = 25 mg/L. The results were expressed in terms of effective loadings. Considering the heterogeneous composition of the test item, Water Accommodated Fractions (WAF) were tested. The results of DOC determinations reflect a rough impression only of exposure conditions over time. No information is available on a molecular weight of the test item and on a potential correlation between effective loadings and measured DOC values.