Ethyl ethyl(phenyl)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:

August 22, 2017 - August 25, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:Room temperature (15-25%)
- Stability under test conditions:Yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 6.25, 12.5, 25, 50 and 100% solutions (nominal concentration).
- Sampling method: Test solutions and control sample were determined at the beginning and at the end of the test. The samples were taken from the test solutions, one from control solution, properly diluted and analysed by an HPLC-UV method.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:A stock solution with a nominal concentration of 100.0 mg/L was prepared with the test item and algal growth medium (OECD Medium) using ultrasonic bath (12 minutes). The test solutions were prepared by the appropriate diluting of this stock solution and distributed into test vessels prior to introduction of algae.
- Controls: (untreated)- Algal growth medium was inoculated with algal cells (without test item)

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY.Cultured under standardised conditions in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd.
- Age of inoculum (at test initiation): 3 days
- Method of cultivation:Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

ACCLIMATION
- Acclimation period: Incubation for 3 days
- Culturing media and conditions (same as test or not):Same as the test
- Any deformed or abnormal cells observed:No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

T=22.7ºC in the flask
T=21.2-23.0ºC in the climate chamber
Temperature checked at the beginning of test and each day thereafter

pH:

pH = 7.32-8.77 (increase not more than 1.5)
pH was checked every day of the beginning of the test, in the control and each concentration

Nominal and measured concentrations:

Nominal:6.25, 12.5, 25.0, 50.0 and 100.0 mg/L.
Measured:6.30, 12.5, 25.3, 50.2 and 100.9 mg/L.
As the measured concentrations deviated not more than 20 per cent from the nominal, biological results are based on the nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel:Erlenmeyer flasks
- Type (delete if not applicable): Closed,covered with air-permeable stoppers
- Material, size, headspace, fill volume: glass, volume 250mL. Fill volume 100mL of test solution
- Aeration: Yes, shaking
- Initial cells density: 104 per mL
- Control end cells density: 70.50 * 104 cell/mL
- No. of vessels per concentration (replicates):3
- No. of vessels per control (replicates):6

GROWTH MEDIUM
- Standard medium used: yes (OECD medium)-reconstituted algae growth medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:Addition of an appropiate volume of different stock solution to deionised water.
- Culture medium different from test medium:No

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH:No
- Photoperiod:Continuous ilumination
- Light intensity and quality:8335 lux(113 uE/m2/s) with fluorescent lamps (400-700 nm) Difference in light density did not exceed +- 15 %.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber. They were determinated at 24, 48 and 72 hours after starts of the test by manual cell counting.
- Chlorophyll measurement: no.
- Other: microscopic observation to detect any abnormalities of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations could be selected for use in the definitive test. Two replicates per each concentration and 3 replicates in the control group were used.
- Test concentrations: 0.1, 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: The significant inhibition was observed at the examined concentration levels during the preliminary range-finding test,therefore five test concentrations in a geometric series with a separation factor of 2.0 and one control were tested in the main experiment.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (Batch Number: A0345704), tested at least twice a year to demonstrate satisfactory test conditions.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 65.14 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf.limit=54.21-78.26

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 21.55 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% conf.limit=19.06-24.36

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 25.79 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% conf.limit=22.70-29.30

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 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.:

ca. 25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Basis for effect:

other: Yield

Remarks on result:

not determinable

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No
- Unusual cell shape:Yes, the chuff , deformed cell was observed at three highest examined cocnentrations of 25, 50 and 100 mgL at 48 and 72 hours after treatment.
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: 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: No
- Effect concentrations exceeding solubility of substance in test medium:No

Results with reference substance (positive control):

- Results with reference substance valid?: YES
- EC50:
72h ErC 50: 0.88 mg/L, (95 % confidence limits: 0.81 – 0.96 mg/L)
72h EyC 50: 0.54 mg/L, (95 % confidence limits: 0.50 – 0.59 mg/L)
72h EbC 50: 0.64 mg/L, (95 % confidence limits: 0.59 – 0.70 mg/L)
These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).The date of the last study (Study Code: 17/058-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 07 – 10 March 2017.

Reported statistics and error estimates:

The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel 2007 for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
The ErC50, EbC50 and EyC50 values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software.
Statistical comparisons of biomass, average specific growth rates and yield in controls and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (alpha = 0.05) by TOXSTAT software.
For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test.

Table No.1:Cell Number (x 10⁴cell/mL) determined in the Main Experiment

NominalConcentration [mg/L]		Number of cells
		0 h	24 h	48 h	72 h
Control		1	4	19	62
		1	5	21	70
		1	4	18	78
		1	3	17	72
		1	4	20	74
		1	3	17	67
	Mean	1.00	3.83	18.67	70.50
	SD	0.0	0.8	1.6	5.6
6.25		1	4	19	66
		1	3	19	62
		1	4	17	60
	Mean	1.00	3.67	18.33	62.67
	SD	0.0	0.6	1.2	3.1
12.5		1	4	15	52
		1	3	17	57
		1	4	14	54
	Mean	1.00	3.67	15.33	54.33
	SD	0.0	0.6	1.5	2.5
25		1	4	10*	28*
		1	4	13*	32*
		1	3	11*	29*
	Mean	1.00	3.67	11.33	29.67
	SD	0.0	0.6	1.5	2.1
50		1	3	7*	13*
		1	3	5*	12*
		1	3	8*	15*
	Mean	1.00	3.00	6.67	13.33
	SD	0.0	0.0	1.5	1.5
100		1	3	3*	5*
		1	3	3*	4*
		1	2	2*	4*
	Mean	1.00	2.67	2.67	4.33
	SD	0.0	0.6	0.6	0.6

Table No.2: Growth Rates (m) and Percentage Inhibition ofmduring the Test Period

NominalConcentration [mg/L]	Growth rate (m) and % inhibition ofm
	0–24 h		0–48 h		0–72 h
	m	%	m	%	m	%
Control	0.0553^+	0.0	0.0609	0.0	0.0591	0.0
6.25	0.0538	2.8	0.0606^+	0.6	0.0575	2.7
12.5	0.0538	2.8	0.0568	6.7	0.0555*	6.1
25.0	0.0538	2.8	0.0505*	17.2	0.0471*	20.3
50.0	0.0458	17.2	0.0391*	35.8	0.0359*	39.2
100.0	0.0401^+	27.4	0.0201*	67.0	0.0203*	65.7

*: statistically significantly different compared to the control values (Bonferroni t-Test;a= 0.05)

+:at these values the rounding of the EXCEL and TOXSTAT software was different. The table contains the values calculated with EXCEL.

Table No.3: Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

NominalConcentration [mg/L]	Area under the Growth Curves (A) and Percentage Inhibition of A
	0–24 h		0–48 h		0–72 h
	A	%	A	%	A	%
Control	34.0	0.0	280.0	0.0	1326.0	0.0
6.25	32.0	5.9	272.0	2.9	1220.0	8.0
12.5	32.0	5.9	236.0	15.7	1048.0*	21.0
25.0	32.0	5.9	188.0*	32.9	656.0*	50.5
50.0	24.0	29.4	116.0*	58.6	332.0*	75.0
100.0	20.0	41.2	60.0*	78.6	120.0*	91.0

*:statistically significantly different compared to the control values (Bonferroni t-Test;alpha= 0.05)

Table No.4:Yield (Y) and Percentage Inhibition of Y during the Test Period

NominalConcentration [mg/L]	Yield (Y) and % inhibition of Y
	0–72 h
	Y	%
Control	69.5	0.0
6.25	61.7*	11.3
12.5	53.3*	23.3
25.0	28.7*	58.8
50.0	12.3*	82.3
100.0	3.3*	95.2

*:statistically significantly different compared to the control values (Bonferroni t-Test;alpha= 0.05)

Although there was a statisticallysignificant differenceat the tested concentration level of 12.5 mg/L (nominal) in case ofgrowth rates, the observed difference(inhibition:m= 6.1 %) is not biologically significant, but is considered to be due to thebiological variability of the test system.

Table No.5: The pH-Values in the Test Media at the Start and End of the Test

n [mg/L]	pH-values
	Start	End
Control	7.32	8.32
		8.26
		8.24
		8.28
		8.31
		8.33
6.25	7.38	8.27
		8.39
		8.33
12.5	7.38	8.24
		8.22
		8.17
25	7.39	8.68
		8.77
		8.65
50	7.40	8.51
		8.43
		8.26
100	7.37	8.14
		8.11
		8.10

Table No.6: Temperature in the Climate Chamber and in the Test Media during the Test

		Exposure Time
		Day 0	Day 1	Day 2	Day 3
temperature (ºC) measured in flask		22.7	22.7	22.7	22.7
min / max temperature (ºC) of the climate chamber	max	23.0	22.9	22.9	22.9
	min	21.7	21.5	21.3	21.2

Validity criteria fulfilled:

yes

Remarks:

1.The cell density in the control cultures increased by the factor of 70.50 in3 days 2.The mean coef.of variation in the control cultures was 14.69 %. 3.The coef. of variation of average specific growth rates during (day 0-3) in control cul. was 1.88 %.

Conclusions:

The EC50(72 h) of the test item in green algae Pseudokirchneriella subcapitata was determinated as 65.14 mg/L (growth rate), NOEC value of 12.5 mg/L and LOEC value of 25 mg/L, both based on growth rate.

Executive summary:

In accordance with OECD TG201 and GLP study, test item was tested to determinated toxicity to aquatic green algae Pseudokirchneriella subcapitata based on growth inhibition. Test was performed in the static condition, during 72 hours. The initial cell density was 1*104 per mL.As first, the preliminary test was performed in the concentrations 0.1, 1, 10, 100 mg/L to determine the approximate toxicity of the test item and that appropriate test concentrations use in the definitive test. The significant inhibition was observed at the examined concentration levels during the preliminary range-finding test,therefore five test concentrations in a geometric series with a separation factor of 2.0 and one control were tested in the main experiment. As the measured concentrations (6.30, 12.5, 25.3, 50.2 and 100.9 mg/L) deviated not more than 20 per cent from the nominal, biological results are based on the nominal concentrations ( 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L). Three replicates at each concentration and six replicates for the untreated controls were performed. For the evaluation of the quality of the algae and validation of the experimental conditions, a positive control is performed with potassium dichromate at least twice a year. The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.All validity criteria were met.The 72h-EC50 of the test item in freshwater algae was determined to be 65.14 mg/L based on growth rate. The NOEC was determined to be 12.5 mg/L and the LOEC with value of 25.0, both based on growth rate. The EC50 (72 h) for the yield was evaluated as 21.55 mg/L, LOEC value was 25.0 mg/L and NOEC value was not possible to measured. The EC50 (72 h) based on biomas was evaluated with value of 25.79 mg/L, NOEC value 6.25 mg/L and LOEC value 12.5 mg/L.

Description of key information

Toxicity to aquatic algae and cyanobacteria

Key study: OECD 201, GLP study. The EC50(72 h) of the test item in green algae Pseudokirchneriella subcapitata was determinated as 65.14  mg/L (growth rate), NOEC value of 12.5 mg/L and LOEC value of 25 mg/L, both based on growth rate.

Key value for chemical safety assessment

EC50 for freshwater algae:

65.14 mg/L

EC10 or NOEC for freshwater algae:

12.5 mg/L

Additional information

Toxicity to aquatic algae and cyanobacteria

Key study: In accordance with OECD TG201 and GLP study, t A freshwater algae growth inhibition test was performed on the test item. Test was performed in the static condition, during 72 hours. The initial cell density was 1*104 per mL. Based on the results obtained from a preliminary range-finding test, where significant inhibition was observed at examined concentration levels, five test concentrations in a geometric series (factor 2.0) and one control were used in the main experiment under static conditions. As the measured concentrations (6.30, 12.5, 25.3, 50.2 and 100.9 mg/L) deviated not more than 20 per cent from the nominal, biological results are based on the nominal concentrations ( 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L). Three replicates at each concentration and six replicates for the untreated controls were performed. A positive control (potassium dichromate) was used to evaluate the quality of the algae and validation of the experimental conditions.All validity criteria were met.The 72h-EC50 of the test item in freshwater algae was determined to be 65.14 mg/L based on growth rate. The NOEC was determined to be 12.5 mg/L and the LOEC with value of 25.0, both based on growth rate. The EC50 (72 h) for the yield was evaluated as 21.55 mg/L, LOEC value was 25.0 mg/L and NOEC value was not possible to measured. The EC50 (72 h) based on biomas was evaluated with value of 25.79 mg/L, NOEC value 6.25 mg/L and LOEC value 12.5 mg/L.