N-ethyl-N-methylcarbamoyl chloride

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

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)

Deviations:

yes

Remarks:

48 hours exposure (validity criteria fulfilled). Results are based on nominal concentrations. However, recovery was 77 and 76% (< 80%)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

48 hours exposure (validity criteria fulfilled). Results are based on nominal concentrations. However, recovery was 77 and 76% (< 80%)

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): EMCA
- Physical state: Colourless liquid
- Analytical purity: 99.3 %

Analytical monitoring:

yes

Remarks:

Due to the nature of the test item, a TOC analysis was performed.

Details on sampling:

The test subtance is extremely unstable in water and immediatly decomposes into ethylmethyl carbamic acid. An analytical method using LC-MS was aattempted in several conditions i.e. basic/acidic/neutral, organic/aqueous, APCI/ESI, positive/negative but it was not possible to develop such analytical method. Therefore a total organic carbon (TOC) analysis was used as a surrogate measurement.

- Concentrations: 100 mg/L and control at time = 0 and t=48 hours, at the end of the exposure period the replicates were pooled at each concentrations before sampling.
- Sampling method: 40mL from the approximate centre of the test vessels.
- Sample storage conditions before analysis: samples were stored in refrigerator until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 23-minutes of magnetic stirring was used to prepare the solution of 100 mg/L. The lower test concentrations were prepared by subsequent dilutions in test medium.
- Controls: 2 control groups with test medium, one with and one without adjustment of the pH value.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No, the test solutions were all clear and colourless.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In house laboratory culture
- Age of inoculum (at test initiation): 4 days before the start of the test, cells from the algal stock culture were inoculated at a cell density of 10000 cells/mL.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from pure culture on agar. The suspensions were continuously aerated and exposed to light in climate room temperature (21-24ºC).

ACCLIMATION
- Acclimation period: 4 days.
- Culturing media and conditions (same as test or not): Not reported.
- Any deformed or abnormal cells observed: No reported.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

24 mg CaCO3/L

Test temperature:

21.7 - 22.4 ºC

pH:

6.7-8

Nominal and measured concentrations:

Nominal concentrations: 0.1, 1.0, 10.0 and 100.0 mg/L
Measured concentrations: see table 5 in 'any other information on results'. The results of the TOC analysis of the samples taken from the highest test concentration and control showed that recoveries for the organic carbon were of 77 % at the start and 76 % after 48 hours of exposure, based on theoretical value (39 % carbon content, based on structural formula) were obtained.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL all-glass
- Material, size, headspace, fill volume: Vessels contained 50 mL of test solution.
- Initial cells density: 10000 cells/mL
- Control end cells density:
Control pH adjusted: 285000
Control: 255000
Two controls were included: test medium and one test medium with pH adjusted to that of the highest concentration (pH-control; pH 6.7).
- No. of vessels per concentration (replicates): 6 replicates for the highest concentration and 3 replicates for the rest of the test item solutions.
- No. of vessels per control (replicates): 6 replicates for the control.

GROWTH MEDIUM
- Standard medium used: according to OECD 201 guideline.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Q water (tap water purified by reverse osmosis =milli-RO, and subsequently passed over activated carbon ion exchange cartridges= Milli-Q water)
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: One control was pH adjusted to that one of the highest concentration at the start of the test (6.7).
- Photoperiod: Continuously
- Light intensity and quality: 30 Watt, 74 to 100 µE/s/m2

EFFECT PARAMETERS MEASURED: Cell densities at 24 and 48 hours.
- Determination of cell concentrations: At the beggining of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometric measurements of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (pathlenght = 20mm). Algal medium was used as blank-

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

growth rate

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

other: Yield

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

growth rate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

other: Yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
- Effect concentrations exceeding solubility of substance in test medium: Tested up to 100 mg/L, the solution was homogeneous, clear and colourless.

Results with reference substance (positive control):

- Results with reference substance valid: Yes
- 72h-EC50: 1.0 mg/L for growth rate inhibition and 0.43 mg/L for yield inhibition.
- Other: It was in accordance with historical data.

Table 1. Mean cell densities (*10000 cells/mL)

Nominal concentration of test item (mg/L)	Exposure time (hours)
	0	24	48
Control, pH adjusted	1.0	6.2	28.5
0.1	1.0	5.8	25.5
1.0	1.0	5.6	25.3
10	1.0	4.6	24.7
100	1.0	6.1	26.1

Table 2. Percentage reduction of growth rate (total test period) and percentage inhibition of yield

Nominal concentration of test substance (mg/L)	Mean growth rate		Yield (0-48 h)
	µ (0-48h)	Reduction (%)	* 10000 cells/mL	Inhibition (%)
Control, pH adjusted	0.06969		27.47
Control	0.06742	3.3	24.55	10.6
0.1	0.06720	3.6	24.25	11.7
1.0	0.06816	2.2	25.73	6.3
10	0.06674	4.2	23.71	13.7
100	0.06791	2.6	25.11	8.6

Table 3. Percentage reduction of growth rate at different time intervals

Nominal concentration of test substance (mg/L)	Mean growth rate
	µ (0-24h)	Reduction	µ (0-48h)	Reduction (%)
Control, pH adjusted	0.07594		0.06344
Control	0.07305	3.8	0.06179	2.6
0.1	0.07152	5.8	0.06288	0.9
1.0	0.07440	2.0	0.06191	2.4
10	0.06311	16.9	0.07037	-10.9
100	0.07489	1.4	0.06093	4.0

Table 4. pH recorded during the final test

Nominal concentration of test item (mg/L)	Exposure time (hours)
	0	48
Control, pH adjusted	8.1	7.9
Control	6.7	7.7
100	6.7	8.0

Table 5. Measured concentrations.

Time of sampling (h)	Sampling date	TOC analysis date	Nominal concentration (mg/l)	Nominal organic carbon conc. (mg/l)	Measured organic carbon conc. (mg/l)	Recovery relative to nominal (%)
0	12/02/2008	26/02/2008	0 100	0 39	1.1 30.1	- 77
48	14/02/2008	26/02/2008	0 100	0 39	1.0 29.7	- 74

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks'. Biomass in controls increased by an average factor > 16 within 48h. Mean section-by-section specific growth rates CV < 35%, average specific growth rates CV < 7% in replicate controls for the whole test period.

Conclusions:

No reduction of growth rate or inhibiton of yield was recorded at any of the concentrations of the test item. The 48 h-NOEC was defined as 100 mg/L and the 48h-EC50 as >100 mg/L.

Executive summary:

A freshwater algae growth inhibition test was performed with the test item according to OECD Guideline 201 under GLP conditions. A combined range-finding / limit test was performed with exponentially growing algal cultures of Pseudokirchneriella subcapitata exposed to nominal test item concentrations of 0, 0.1, 1, 10 and 100 mg/L. Two controls were included i.e. one with and one without pH adjustment. 6 replicates were tested for the control and limit (100 mg/L) concentrations, whereas 3 replicates were tested for the 0.1, 1.0 and 10 mg/L test item concentrations. Exposure was 48 hours under static conditions, samples for analytical determination were taken at start and at the end of the test. Due to the highly reactive nature of the test item with water, a conventional chromatography-based method for the analytical determination of the concentration was not possible. Nevertheless, a TOC analysis was used as a surrogate analytical support. Recoveries of 77% at the start and 76% and the end of the test were obtained based on theoretical value. The study met the acceptability criteria. No reduction of growth rate or inhibiton of yield was recorded at any of the concentrations. The 48 h-NOEC was defined as 100 mg/L and the 48h-EC50 as >100 mg/L.

Description of key information

Key study: Test method according to OECD 201. GLP study. The 48h-EC50 was > 100 mg/L and the NOEC was 100 mg/L based on growth inhibition rate.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

Additional information

Key study: A freshwater algae growth inhibition test was performed with the test item, according to OECD Guideline 201 under GLP conditions. A combined range-finding limit test was performed under static conditions with exponentially growing algal cultures of Pseudokirchneriella subcapitata exposed for 48h to nominal test item concentrations of 0, 0.1, 1, 10 and 100 mg/L. No reduction of growth rate or inhibiton of yield was recorded at any of the concentrations. The 48 h-NOEC was defined as 100 mg/L and the 48h-EC50 as >100 mg/L based on nominal concentration.