Carbamic acid, N-[(butylthio)thioxomethyl]-, butyl ester

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 July - 8 August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also in accordance with GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

(2006)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

(2008)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 8692: "Water quality - Fresh water algal growth inhibition test with unicellular green algae" (2012)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23 (2000)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

butyl N-[(butylsulfanyl)methanethioyl]carbamate

EC Number:

691-061-8

Cas Number:

1001320-38-2

Molecular formula:

C10H19NO2S2

IUPAC Name:

butyl N-[(butylsulfanyl)methanethioyl]carbamate

Test material form:

solid

Remarks:

yellow grease-like solid

Specific details on test material used for the study:

Molecular formula: C10H19NO2S2
Name: n-butoxycarbonyl n-butyl dithiocarbamate (active ingredient)
CAS Number: 1001320-38-2

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations
Range finding test: 1, 10 and 100 mg/L water accommodated fraction (WAFs)
Final test: blank control, 1.0, 3.2, 10, 32 and 100% of a WAF prepared at a loading rate of 1.0 mg/L and 100% of a WAF prepared at a loading rate of 3.2 mg/L
- Sampling method
Frequency: at t=0 h, t=24 h and t=72 h
Volume: 2.0 mL
- Sample storage conditions before analysis: Samples not analysed on the day of sampling were stored in a freezer until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance was warmed to approximately 28°C in order to melt. The liquid test substance was used for weighing out the required amounts of test substance. All loading rates of 1.0 mg/L and higher were prepared separately applying one day of magnetic stirring to reach maximum solubility of the test substance in the test medium. The resulting aqueous mixtures were left to stabilize for 45-55 minutes where after the clear and colourless WAFs were taken out by siphoning. The lower test concentrations for the final test were prepared by subsequent dilutions of the 1.0 mg/L WAF in test medium. After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. No correction was made for the purity/composition of the test substance.
- Controls: Test medium without test substance or other additives

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Species: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Method of cultivation:
Algae stock cultures were started by inoculating growth medium (M1) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Acclimation period: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2, according to OECD 201) at a cell density of 1 x 10^4 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

The temperature of the test medium was 23.1°C at the start of the test. During the exposure period the temperature was maintained between 22.0 and 23.1°C.

pH:

Start: 8.0-8.1
End: 7.8-7.9

Nominal and measured concentrations:

- Nominal
Final test: blank control, 1.0, 3.2, 10, 32 and 100% of a WAF prepared at a loading rate of 1.0 mg/L and 100% of a WAF prepared at a loading rate of 3.2 mg/L.

- Measured: See "any other information on results incl. tables".

Details on test conditions:

TEST SYSTEM (final test)
- Test vessel: All glass with a capacity of 100 mL and containing 50 mL of test solution
- Type: Closed
- Initial cells density: 1x10^4 cells/ml
- No. of vessels per concentration (replicates): 3 replicates
- No. of vessels per control (replicates): 6 replicates

GROWTH MEDIUM
- Standard medium used: yes, M2 prepared according to OECD 201.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap-water purified by reverse osmosis)
- Culture medium different from test medium: Yes (M1). Three days before the start of the test the algal stock culture were inoculated in the same culture medium (M2) used in the test. The culture was maintained under the same conditions as used in the test.
- Intervals of measurements: pH was measured at the beginning and at the end of the test. Temperature was continuously measured in a control vessel. At the end of the final test microscopic observations were performed on all test concentrations to observe for any abnormal appearance of the algae.

OTHER TEST CONDITIONS
- Photoperiod: Continuous
- Light intensity and quality: TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 89 to 92 μE.m-2.s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
- Determination of cell concentrations: Cells were counted using a microscope and a counting chamber.

TEST CONCENTRATIONS
- Combined limit/range-finding test concentrations: blank control, WAFs prepared at a loading rate of 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes
- Spacing factor for test concentrations in the final test: approximately 3x

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.82 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (95% CL: 0.25-2.7 mg/L)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.21 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.11 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Combined limit/range-finding test: The expected EC50 for growth rate reduction was between TWA concentrations of 0.60 and 7.1 mg/L. The expected EC50 for yield inhibition was below a TWA concentration of 0.60 mg/L. From 48 hours of exposure onwards a floating layer was observed at the 100 mg/L WAF.


- Final test
Measured concentrations: Test concentrations did not remain stable during the test period, i.e. they decreased to either below the LOD (0.014 mg/L) or to 5-79% of initial at the end of the test. Consequently, effect parameters were based on Time Weighted Average (TWA) concentrations.

Biological results: Growth rates were in the range of the controls at TWA concentrations from 0.0070 to 0.11 mg/L during the 72-hour test period, whereas the growth rate of algae exposed to 0.51 mg/L TWA and higher were increasingly reduced. Statistically significant reduction of growth rate was found at test concentrations of 0.51 mg/L TWA and higher. Inhibition of yield increased with increasing concentration of S-10903 from 0.51 mg/L TWA upwards resulting in 100% inhibition at 2.6 mg/L TWA. Statistically significant inhibition of yield was found at test concentrations of 0.51 mg/L TWA and higher.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

- Acceptability of the test:
1. In the control, cell density increased by an average factor of >16 within two days.
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35%.
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7%.

Results with reference substance (positive control):

- Potassium dichromate: EC50 for growth rate reduction (ERC50: 0-72h) was 1.1 mg/L (95% confidence levels ranging from 0.71 to 1.6 mg/L). The sensitivity of this batch of Pseudokirchneriella subcapitata was within the range of the historical data collected at WIL Research Europe.

Any other information on results incl. tables

Measured concentrations versus loading rates or percentages thereof
Loading rate (mg/L)^1)	Measured concentration (mg/L)			TWA conc. (mg/L)
	t=0 h	t=24 h	t =72h
1.0% of 1.0	<0.014^2	<0.014^2	<0.014^2	0.007
3.2% of 1.0	0.027	<0.014^2	<0.014^2	0.0093
10% of 1.0	0.0812	0.025	<0.014^2	0.024
32% of 1.0	0.279	0.19	0.014	0.11
1	0.871	0.645	0.231	0.51
3.2	2.88	2.66	2.28	2.6
^1WAF prepared at the given loading rate
^2Values below the Limit of Detection (LOD was 0.014 mg/L) were used in calculations as being half of this value.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test material significantly reduced the growth rate and inhibited the yield of Pseudokirchneriella subcapitata at a TWA concentration of 0.51 mg/L and higher. The EC50 for growth rate reduction (72h-ErC50) was 0.82 mg/L (95% confidence level ranging from 0.25 to 2.7 mg/L). The 72h-NOEC for growth rate reduction was 0.11 mg/L.

Executive summary:

In a 72 h toxicity study conducted according to OECD guideline 201 and following GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to S-10903 at the following nominal concentrations: 0 (blank-control), 1.0, 3.2, 10, 32 and 100% of a WAF prepared at a loading rate of 1.0 mg/L and 100% of a WAF prepared at a loading rate of 3.2 mg/L. The EC50 for growth rate reduction (72h-ErC50) was 0.82 mg/L (95% confidence levels 0.25 - 2.7mg/L), the 72h-ErC10 was 0.21 mg/L (95% confidence level 0.061 - 0.71 mg/L) and the 72h-NOEC for growth rate reduction was 0.11 mg/L, based on TWA concentrations. The study is considered to be reliable without restrictions