N-carbamoylmethyliminodi(acetic acid)

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:

2017-01-09 to 2017-09-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Adopted 23rd March 2006, Annex 5 corrected: 28th July 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

March 01, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L

- Sampling method: For determination of the test item concentrations from the control one sample, from the treatment groups three replicate samples were taken (1 mL/replicate). The samples were directly injected and analysed by the above detailed HPLC method.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test solutions used in the test were prepared by mechanical dispersion. A stock solution was first prepared by dissolving an amount of 0.08 g test item in 800 mL dilution water (OECD medium) resulting a nominal concentration of 100 mg/L. This stock solution was placed into the ultrasonic bath for appr. 5 minutes thereafter the pH level of the stock solution was adjusted with NaOH to the pH level of the OECD medium. The test solutions of subsequent lower test concentrations were prepared by appropriate diluting of this stock solution. The test solutions were prepared just before introduction of algae (start of the experiment).

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
- Source: Collection of Algal Cultures, 37073 Göttingen, Germany
- Method of cultivation: algae were kept as stock culture on solid agar

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Hardness:

no data

Test temperature:

22.0 - 22.2°C (measured in the flasks);
21.0 - 23.1°C (in the climate chamber)

pH:

8.18– 8.96 (in the control group);
7.84-8.95 (in the treatment groups)

Dissolved oxygen:

not specified

Salinity:

not applicable

Conductivity:

not specified

Nominal and measured concentrations:

Nominal concentration: 6.25, 12.5, 25, 50 and 100 mg/L;
Measured concentration: The recovery rate of the five concentration was 94 % - 111 % at the start and 90% - 108 % after 72 hours.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks 250 mL
- Type: air-permeable stoppers
- Initial cells density: 10000 cells/mL
- Control end cells density: approx. 10000000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted algal growth medium (acc. to OECD TG 201)


OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: continuous
- Light intensity and quality: 8006 Lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: manual counting at 24, 48 and 72 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Yes
- Test concentrations: 6.25, 12.5, 25, 50 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities none reported
- Effect concentrations exceeding solubility of substance in test medium: no
- Mean coefficient of variation for section-by-section specific growth rates: 5.90 % (i.e. < 35 %)

Results with reference substance (positive control):

- Results with reference substance valid? YES
- The 72h ErC50: 0.71 mg/L, (95 % confidence limits: 0.66 – 0.77 mg/L)
- The 72h EyC50: 0.46 mg/L, (95 % confidence limits: 0.42 – 0.49 mg/L)

Validity of the Test

- The cell density in the control cultures increased by a factor of 85.0 within 72 hours. This corresponds to a specific growth rate of 1.48 per day.

- The mean coefficient of variation (CV) for section-by-section specific growth rates (days 0 - 1, 1 - 2 and 2 - 3, for 72 h-tests) in the control cultures did not exceed 35 %.

- CV for section-by-section growth rate day 0-1: 6.40 %

- CV for section-by-section growth rate day 1-2: 5.70 %

- CV for section-by-section growth rate day 2-3: 5.61 %

The mean coefficient of variation for section-by-section specific growth rates: 5.90 %.

- The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 1.21 %.

 

Analytical Results

Five different concentrations arranged in a geometric series with a spacing factor of 2 and a concurrent control was tested in the main experiment. The nominal test item concentrations were 6.25, 12.5, 25, 50 and 100 mg/L. The measured concentrations of the test item varied between 94 and 111 % of the nominal at the start and between 90 % and 108 % at the end of the experiment. In the untreated control group the test item was not detected. Therefore, the biological results were based on the nominal concentrations.

Table 1: ADA concentrations measured in the test solutions

Nominal Concentration, mg/L	Measured concentrations with 95% confidence interval	In percentage of the nominal	Measured concentrations with 95% confidence interval	In percentage of the nominal
6.25	6.37 ± 0.31 mg/L	102%	5.63 ± 0.14 mg/L	90%
12.5	11.8 ± 0.11 mg/L	95%	11.4 ± 0.76 mg/L	92%
25	23.6 ± 0.05 mg/L	94%	22.8 ± 0.05 mg/L	91%
50	55.3 ± 0.2 mg/L	111%	54.2 ± 0.1 mg/L	108%
100	95.2 ± 0.1 mg/L	95%	94.1* mg/L	94%

* Only two replicate samples were evaluated because of an analytical error

 

Biological Results

Based on the results of the statistical evaluation (Dunnett’s-Test (α = 0.05; 2-Tailed) by SPSS software) neither the average specific growth rate nor the yield showed significant difference compared to the control in any of the treated concentrations. Accordingly, the 72-h LOEC and EC₅₀ values based on average specific growth rate and yield were determined to be > 100 mg/L.

Table 2: Summary of the biological endpoints

Endpoints (0-72 h)	Growth rate (µ)	Yield (y)
EC10	> 100 mg/L	> 100 mg/L
EC20	> 100 mg/L	> 100 mg/L
EC50	> 100 mg/L	> 100 mg/L
NOEC	100 mg/L	100 mg/L
LOEC	> 100 mg/L	> 100 mg/L

 

Table 3: Algal Cell Concentrations (104 cell/ mL) during the Test Period of 72 Hours and Yield

Concentration [mg/L]	Number of Cells (x 10^4/mL)
Control		0 h	24 h	48 h	72 h	Yield
		1	4	15	89	88
		1	4	16	85	84
		1	5	17	89	88
		1	4	16	80	79
		1	4	17	79	78
		1	4	15	88	87
	Mean	1.00	4.17	16.00	85.00	84.00
	SD	0.00	0.41	0.89	4.52	4,52
6.25		1	4	19	89	88
		1	5	18	81	80
		1	3	18	89	88
	Mean	1.00	4.00	18.33	86.33	85.33
	SD	0.00	1.00	0.58	4.62	4,62
12.5		1	4	18	88	87
		1	4	18	86	85
		1	4	19	81	80
	Mean	1.00	4.00	18.33	85.00	84.00
	SD	0.00	0.00	0.58	3.61	3,61
25		1	4	17	85	84
		1	3	18	88	87
		1	5	18	83	82
	Mean	1.00	4.00	17.67	85.33	84.33
	SD	0.00	1.00	0.58	2.52	2,52
50		1	3	18	88	87
		1	3	17	88	87
		1	4	18	89	88
	Mean	1.00	3.33	17.67	88.33	87.33
	SD	0.00	0.58	0.58	0.58	0,58
100		1	2	17	81	80
		1	3	18	78	77
		1	3	18	84	83
	Mean	1.00	2.67	17.67	81.00	80.00
	SD	0.00	0.58	0.58	3.00	3,00

 

Table 4: The Specific Growth Rates during the Test Period of 72 Hours

Concentration [mg/L]	Growth Rates (µ)
		0 - 24 h	0 - 48 h	0 - 72 h
Control		0.0578	0.0564	0.0623
		0.0578	0.0578	0.0617
		0.0671	0.0590	0.0623
		0.0578	0.0578	0.0609
		0.0578	0.0590	0.0607
		0.0578	0.0564	0.0622
	Mean	0.0593	0.0577	0.0617
	SD	0.0038	0.0012	0.0007
6.25		0.0578	0.0613	0.0623
		0.0671	0.0602	0.0610
		0.0458	0.0602	0.0623
	Mean	0.0569	0.0606	0.0619
	SD	0.0107	0.0007	0.0008
12.5		0.0578	0.0602	0.0622
		0.0578	0.0602	0.0619
		0.0578	0.0613	0.0610
	Mean	0.0578	0.0606	0.0617
	SD	0.0000	0.0007	0.0006
25		0.0578	0.0590	0.0617
		0.0458	0.0602	0.0622
		0.0671	0.0602	0.0614
	Mean	0.0569	0.0598	0.0618
	SD	0.0107	0.0007	0.0004
50		0.0458	0.0602	0.0622
		0.0458	0.0590	0.0622
		0.0578	0.0602	0.0623
	Mean	0.0498	0.0598	0.0622
	SD	0.0069	0.0007	0.0001
100		0.0289	0.0590	0.0610
		0.0458	0.0602	0.0605
		0.0458	0.0602	0.0615
	Mean	0.0401	0.0598	0.0610
	SD	0.0098	0.0007	0.0005

 

Table 5: Growth Rates (µ) and Percentage Inhibition of µ during the Test Period

Concentration [mg/L]	Growth Rates (µ) and % Inhibition of µ
	0 – 24 h		0 – 48 h		0 – 72 h
	µ	%	µ	%	µ	%
Control	0.0593	0.0	0.0577	0.0	0.0617	0.0
6.25	0.0569	4.1	0.0606	-4.9	0.0619	-0.4
12.5	0.0578	2.6	0.0606	-4.9	0.0617	0.0
25	0.0569	4.1	0.0598	-3.6	0.0618	-0.1
50	0.0498	16.1	0.0598	-3.6	0.0622	-0.9
100	0.0401	32.3	0.0598	-3.6	0.0610	1.1

 

Table 6: Yield (Y) and Percentage Inhibition of Y during the Test Period

Concentration [mg/L]	Yield y and % Inhibition of y 0 - 72 h
	Y	%
Control	84.00	0.0
6.25	85.33	-1.6
12.5	84.00	0.0
25	84.33	-0.4
50	87.33	-4.0
100	80.00	4.8

 

Table 7: Results of the statistical evaluation based on the Yield and Growth rate (Dunnett’s Test)

Dependent Variable	(mg/L)	Mean Difference compared to the Control	Std. Err	Sig.	95% Confidence Interval
					Lower Bound	Upper Bound
Growth rate	6.25	0.0001833	0.0004222	0.933	-0.001028	0.001395
	12.50	-0.0000167	0.0004222	1.000	-0.001195	0.001228
	25.00	-0.0000833	0.0004222	1.000	-0.001128	0.001295
	50.00	0.0005500	0.0004222	0.624	-0.000661	0.001761
	100.00	-0.0006833	0.0004222	0.425	-0.001895	0.000528
Yield	6.25	1.33333	2.59487	0.985	-6.1110	8.7777
	12.50	0.00000	2.59487	1.000	-7.4444	7.4444
	25.00	0.33333	2.59487	1.000	-7.1110	7.7777
	50.00	3.33333	2.59487	0.636	-4.1110	10.7777
	100.00	-4.00000	2.59487	0.470	-11.4444	3.4444

 

Table 8: The section-by-section specific growth rates in the control cultures

Section-by-Section Growth Rate
Expressed in hours	0–24 h	24-48 h	48-72 h
	0.0593	0.0562	0.0696
Expressed in days	0-1 day	1-2 day	2-3 day
	1.4235	1.3856	1.4805

 

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the obtained results showed that the test item had no toxic effect up to a concentration of 100 mg/L (i.e. limit test concentration) on the growth of green algae; the EC10, EC20, EC50 results and the LOEC are higher than 100 mg/L.

Executive summary:

The effect of the test item on algal growth was assessed using the unicellular green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum) in a GLP-study according to OECD TG 201. For this purpose, exponentially-growing cultures of Raphidocelis subcapitata were exposed to the test item over several generations under defined conditions (static test). The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included three replicates per test concentration and six replicates in control. The alga cell concentration was approximately 10e4 cells/mL at the start of the test for all of the test cultures. Glass flasks with a total capacity of 250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The alga cell concentration was determined by microscope in each testing flask at 24, 48 and 72 hours. In the preliminary range finding tests on algae, the test item showed slight toxic effect at 100 mg/L concentration therefore the main test was performed at 100, 50, 25, 12.5, 6.25 mg/L concentrations (with pH adjustment of the test item stock solution) including a concurrent control. The purpose of the analytical part of the study was to verify the concentrations of the test item in the test medium. The measured test item concentration remained within ± 20 % of the nominal over the test period of 72 hours, therefore the biological results are based on the nominal concentrations. Statistical comparisons of average specific growth rates and yield in control and in treated group were carried out using ANOVA with Dunnett’s Test (α = 0.05, SPSS software). The results of the statistical evaluation showed that the 0 - 72 h average specific growth rate and yield in the treatment group (100 mg/L) were not statistically significantly different from the untreated concurrent control. All validity criteria were met. The No Observed Effect Concentration (NOEC) was determined to be 100 mg/L and the Lowest Observed Effect Concentration (LOEC) was determined to be > 100 mg/L (limit concentration). As the test item had no significant effect on the growth of algae therefore statistical analysis was not performed to determine the EC values. Consequently, the 72-h EC₅₀ values based on growth rate and yield were also > 100 mg/L. All biological results are based on the nominal test item concentrations.

Description of key information

Under the test conditions, the obtained results showed that the test item had no toxic effect up to a concentration of 100 mg/L (i.e. limit test concentration) on the growth of green algae; the EC₁₀, EC₂₀, EC₅₀ results and the LOEC are higher than 100 mg/L (reference 6.1.5-1).

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The effect of the test item on algal growth was assessed using the unicellular green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum) in a GLP-study according to OECD TG 201. For this purpose, exponentially-growing cultures of Raphidocelis subcapitata were exposed to the test item over several generations under defined conditions (static test). The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included three replicates per test concentration and six replicates in control. The alga cell concentration was approximately 10e4 cells/mL at the start of the test for all of the test cultures. Glass flasks with a total capacity of 250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The alga cell concentration was determined by microscope in each testing flask at 24, 48 and 72 hours. In the preliminary range finding tests on algae, the test item showed slight toxic effect at 100 mg/L concentration therefore the main test was performed at 100, 50, 25, 12.5, 6.25 mg/L concentrations (with pH adjustment of the test item stock solution) including a concurrent control. The purpose of the analytical part of the study was to verify the concentrations of the test item in the test medium. The measured test item concentration remained within ± 20 % of the nominal over the test period of 72 hours, therefore the biological results are based on the nominal concentrations. Statistical comparisons of average specific growth rates and yield in control and in treated group were carried out using ANOVA with Dunnett’s Test (α = 0.05, SPSS software). The results of the statistical evaluation showed that the 0 - 72 h average specific growth rate and yield in the treatment group (100 mg/L) were not statistically significantly different from the untreated concurrent control. All validity criteria were met. The No Observed Effect Concentration (NOEC) was determined to be 100 mg/L and the Lowest Observed Effect Concentration (LOEC) was determined to be > 100 mg/L (limit concentration). As the test item had no significant effect on the growth of algae therefore statistical analysis was not performed to determine the EC values. Consequently, the 72-h EC₅₀ values based on growth rate and yield were also > 100 mg/L. All biological results are based on the nominal test item concentrations.