1,3-bis(hydroxymethyl)urea

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 2011-11-02 to 2012-07-19

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Sampling was performed at the start of the exposure (0 h) from the inoculated (+ algae) replicate 7 of each concentration and the control. At the end of the exposure (72 h) samples were taken from the combined inoculated replicates of the test concentrations and of the combined inoculated control group replicates.
Additionally, samples were collected from the uninoculated (- algae) replicate 0 of each test concentration and the control at 24, 48 and 72 hours to determine the stability of the test substance under test conditions.

Vehicle:

no

Details on test solutions:

The stock test solutions were prepared by directly adding test substance to 1L of test medium and stirring for approximately 10 min at 20 ± 2 °C until visibly dissolved. The stock test solutions were prepared at 111% of the nominal concentration to compensate for the dilution by the later addition of algal inoculum. To reach the correct nominal concentrations at test start, inoculum culture was added to each 111% stock test solution at a ratio of 1:10.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green Algae
- Strain: CHODAT SAG 86.81
- Source: SAG, Collection of algal cultures in University of Göttingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: An inoculum culture in exponential growth phase was prepared with an aliquot of the stock algal culture added to sterile test media to provide an initial cell density of 0.5 x 104 cells/mL.

ACCLIMATION
- Acclimation period: incubated for 4 days at 21 – 24 °C
- Culturing media and conditions: same as test
- Inoculum culture morphology: normal and healthy

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.9 – 23.1 °C

pH:

7.9 - 8.6

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 1.0, 3.2, 10, 32, 100 mg/L based on test substance mass without correction for purity
Mean measured concentrations: 0 (control), corresponding to 0.86, 2.7, 8.1, 27.1, 83.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (nominal volume 250 mL) plugged with gas permeable silicone sponge caps
- Type: closed
- Material, size, headspace, fill volume: 100 mL (inoculated); 200 mL (uninoculated)
- Initial cells density: 0.5 x 104 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: test medium according to OECD 201

OTHER TEST CONDITIONS
- Shaking rate: Continuous (approx. 85 rpm)
- Adjustment of pH: no
- Photoperiod: permanent illumination
- Light intensity and quality: Artificial light, Average 6993 lux at a wave length of 400 - 700 nm

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

23.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 20.9 – 25.5 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

58 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 55.4 – 60.8 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

12.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 10.8 – 15.4 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 26.4 – 29.6 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

8.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

27.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

yield

Results with reference substance (positive control):

The ErC50 (72 h) of the control substance potassium dichromate was 0.87 mg/L (26 Sep 2011) which is in the range recommended in the ISO 8692 guideline: ErC50 = 0.71 – 0.97 mg/L after 72 hours for Desmodesmus subspicatus.

VALIDITY CRITERIA

This test was fully compliant with all the following validity criteria required by the corresponding test guidelines and is considered valid.

• The validity criterion for cell multiplication factor in the untreated control is >16 in 72 hours. The cell multiplication factor in the untreated control was 100-fold after 72 hours (microscopically counted cell density: 50 x 10⁴ cells/mL).

• The validity criterion for the mean coefficient of variation for section-by-section growth rates for each test day in the control cultures is ≤35 %. The mean coefficient of variation for section-by-section growth rates for each test day in the control cultures was 12.9 %.

• The validity criterion for the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures is ≤7 %. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 1.4 %.

ANALYTICAL CONCENTRATION CONTROL

Method of analysis: RP - High performance liquid chromatography with UV detection

Limit of quantification (LOQ): 1 mg/L

Recovery efficiency: 100 – 104 %

Table 1. Analytically measured concentrations of the test substance in the test solutions

Nominal concentration [mg/L]	Analytically measured concentrations				Geometric mean measured concentration [mg/L]	Mean measured concentration [% of nominal]
	0 hour test solution		72 hour test solution
	[mg/L]	[% of nominal]	[mg/L]	[% of nominal]
0	n.d.	-	n.d.	-	-	-
1	1.1	110%	<1	-	0.86^[a]	86%
3.2	3.2	100%	2.1	66%	2.6	81%
10	9.9	99%	6.7	67%	8.1	81%
32	32.0	100%	23	72%	27.1	85%
100	97.0	97%	69	69%	81.8	82%

[a] The 72-h concentration was estimated as 0.86 mg/L, reflecting the average loss in the other test groups, in order to calculate the mean.

 

The measured concentrations of the test substance in the test water were in a range of 100 + 20% of the nominal concentration in all tested concentrations at the start of exposure and decreased to 66 – 72 % of the nominal concentration at the end of exposure. However, all measured concentrations were within a range of + 20% of the mean measured concentration over the whole exposure period.

 

Table 2. 72-h Effect concentrations (mg/L) based on mean measured concentration

Algal Yield [mg/L]		Algal Growth Rate [mg/L]
EyC10 confidence limits 95%:	12.9 10.8 – 15.4	ErC10 confidence limits 95%:	23.1 20.9 – 25.5
EyC20 confidence limits 95%:	16.8 14.9 – 19.0	ErC20 confidence limits 95%:	31.7 29.4 – 34.1
EyC50 confidence limits 95%:	28.0 26.4 – 29.6	ErC50 confidence limits 95%:	58.0 55.4 – 60.8
NOEyC	8.10	NOErC	8.10
LOEyC	27.1	LOErC	27.1

 

Validity criteria fulfilled:

yes

Conclusions:

In conclusion the 72 hour effect concentrations based on mean measured concentration for the test item in Desmodesmus subspicatus are ErC10 = 23.1 mg/L, ErC50 = 58.0 mg/L and NOEC = 8.10 mg/L.

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to OECD 201, EC 440/2008 C.3 and under GLP. The study was conducted under static conditions with an initial cell density of 0.5 x 10⁴ cells/mL for a period of 72 hours. Five nominal concentrations were tested in a geometrical series: 1, 3.2, 10, 32 and 100 mg/L. The measured concentrations of the test item in the fresh media (0 h) were in the range of 97 to 110 % of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 66 to 72 % of the nominal values of all concentrations. However, all measured concentrations were within a range of + 20 % of the mean measured concentration over the whole exposure period. Therefore, all effect values given are based on the mean measured test item concentrations. In this study, the test item was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours with the following effect values: The EC50-values with 95 % confidence interval for inhibition of growth rate (ErC50) was 58.0 mg/L (55.4 – 60.8 mg/L) and for inhibition of yield (EyC50) was 28.0 mg/L. The EC10 value with 95 % confidence interval was determined to be 23.1 mg/L (20.9 – 25.5 mg/L) based on growth rate (ErC10) and 12.9 mg/L based on yield (EyC10).

Description of key information

The 72 -hour effect concentrations based on mean measured concentration for the test item in Desmodesmus subspicatus are ErC10 = 23.1 mg/L, ErC50 = 58.0 mg/L and NOEC = 8.10 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

58 mg/L

EC10 or NOEC for freshwater algae:

23.1 mg/L

Additional information

 The toxicity of the test item to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to OECD 201, EC 440/2008 C.3 and under GLP. The study (reference 6.1.5-1) was conducted under static conditions with an initial cell density of 0.5 x 10⁴ cells/mL for a period of 72 hours. Five nominal concentrations were tested in a geometrical series: 1, 3.2, 10, 32 and 100 mg/L. The measured concentrations of the test item in the fresh media (0 h) were in the range of 97 to 110 % of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 66 to 72 % of the nominal values of all concentrations. However, all measured concentrations were within a range of + 20 % of the mean measured concentration over the whole exposure period. Therefore, all effect values given are based on the mean measured test item concentrations. In this study, the test item was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours with the following effect values: The EC50-values with 95 % confidence interval for inhibition of growth rate (ErC50) was 58.0 mg/L (55.4 – 60.8 mg/L) and for inhibition of yield (EyC50) was 28.0 mg/L. The EC10 value with 95 % confidence interval was determined to be 23.1 mg/L (20.9 – 25.5 mg/L) based on growth rate (ErC10) and 12.9 mg/L based on yield (EyC10).