Fatty acids, tall-oil, polymers with glycidyl neodecanoate

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 August 11, 2015 to December 1, 2015

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:

yes

Remarks:

The measured start cell concentration was higher than stated in the guideline. As all validity criteria were met and exponential growth was observed in the control this was stated as uncritical.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

The measured start cell concentration was higher than stated in the guideline. As all validity criteria were met and exponential growth was observed in the control this was stated as uncritical.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

At 0 and 72 h

Details on test solutions:

PREPARATION OF TEST SOLUTION
Before the experiment, the water-accommodated fractions (WAF) of the concentrations to be tested were prepared. This was done by weighing the nominal loads, (real loads 10.9, 18.1, 32.2, 57.1 and 100.7 mg/L) adding the appropriate amount of nutrient medium (demineralised water enriched with minerals but without algae) and shaking for 23 h. After membrane filtration, the solutions were used to prepare the treatments.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Unicellular freshwater green alga.
- SAG strain number: 86.81.
- Taxonomic position: Chlorophyta - Chlorophyceae.

ORIGIN AND CULTURE
The culture of Desmodesmus subspicatus was obtained in February 2014 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8°C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

IN LIFE DATES: From August 31, 2015 to September 04, 2015.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

20.8 – 23.2°C

pH:

Blank control: 7.7 - 9.0
Treatment groups: 7.6 - 8.8

Nominal and measured concentrations:

0, 10, 18, 32, 56 and 100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass flasks total volume 65 mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per blank control (replicates): 6

OTHER TEST CONDITIONS
- Light intensity and quality: 5300 Lux.

PARAMETERS CALCULATED
- Cell numbers
- Growth rate
- Yield
- Inhibition

TEST CONCENTRATIONS
- Test concentrations: 10, 18, 32, 56 and 100 mg/L (nominal concentrations).
- Blank control: Deionised water with nutrient medium and algae.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate in a separate study (GLP study no. 15011201R301)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

56 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:

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:

NOEC

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC50

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 (for algal test): Yes

Results with reference substance (positive control):

72h ErC50: 1.1 mg/L (95% confidence interval- 0.95 – 1.3 mg/L)
72h EyC50: 0.39 mg/L (95% confidence interval- 0.33 – 0.44 mg/L)

FINDINGS

Cell Numbers

The cell numbers were determined by photometric measurement of optical density. The means and standard deviations of the cell numbers of the control and the treatments are presented in the following table:

Table 1: Cell number/mL

Nominal concentration in mg/L	Parameter	Cell number/mL
		0 h	24 h	48 h	72 h
Blank control	Mean	6468	20662	106003	551577
	SD	0	3369	9265	48391
10	Mean	6468	21560	99176	510613
	SD	0	4312	13507	56452
18	Mean	6468	22997	87677	452760
	SD	0	622	10246	70169
32	Mean	6468	30184	89474	504504
	SD	0	2852	9581	54703
56	Mean	6468	18685	88755	476835
	SD	0	3465	6587	79263
100	Mean	6468	16529	78335	439105
	SD	0	2713	7178	35579

 

Microscopical Observations

The appearance of the algae at the end of the test was normal and healthy in blank control as well as treatment groups.

Growth Rate and yield

From the cell numbers, the Growth rate μ and the yield were calculated. The means and standard deviations at the end of the test are given in the following table:

Table 2: Growth Rate μ and the Yield

Nominal concentration in mg/L		Growth rate (0 - 72 h) [d^-1]	Yield (0 - 72 h) [cell concentration/mL]
Blank control	Mean	1.48	545109
	SD	0.03	48391
10	Mean	1.46	504145
	SD	0.04	56452
18	Mean	1.41	446292
	SD	0.05	70170
32	Mean	1.45	498036
	SD	0.04	54703
56	Mean	1.43	470367
	SD	0.06	79263
100	Mean	1.41	432637
	SD	0.03	35579

 

Inhibition

The following mean inhibition values were calculated for the growth rate μ and the yield.

Table 3: Inhibition values

Nominal concentration in mg/L	% Inhibition
	Growth rate (0 – 72 h)	Yield (0 - 72 h)
Blank control	0	0
10	1.76	7.51
18	4.55	18.13
32	2.02	8.64
56	3.43	13.71
100	5.11	20.63

VALIDITY CRITERIA

Growth

- Increase factor: The cell concentration in the blank control should increase by a factor of at least 16 within 72 h.

- Daily growth rates: Mean coefficient of variation of daily growth rates in the blank controls should be 35% at the most. Coefficient of variation of average growth rate during the whole test period should be 7% at the most.

- Values and assessment: The daily growth rates of the blank controls were calculated. Means, standard deviations and coefficients of variation were determined. Values and assessment can be found in the following tables:

Table 4: Daily Growth Rates of the Controls

Growth rates	Days 0 – 1	Days 1 – 2	Days 2 – 3	CV of sectional daily growth rates	Days 0 – 3
Replicate 1	1.099	1.528	1.668	21%	1.43
Replicate 2	1.386	1.365	1.630	10%	1.46
Replicate 3	0.981	1.833	1.666	30%	1.49
Replicate 4	1.299	1.572	1.577	11%	1.48
Replicate 5	1.041	1.802	1.673	27%	1.51
Replicate 6	1.099	1.754	1.682	24%	1.51
Mean	1.151	1.642	1.649	20%	1.481
Standard deviation	0.157	0.184	0.040		0.030
Coefficient of variation (CV)	14%	11%	2%		2%

 

Table 5: Assessment

Parameter	Validity criteria	Observed value	Assessment
Increase factor biomass	factor 16 in 72 h	85	Valid
Mean coefficient of variation ofdaily growth rates	maximum 35%	20%	Valid
Coefficient of variation of average growth rate during the whole test period	maximum 7%	2%	Valid

 

Change of pH

The pH of the blank control should not fluctuate by more than 1.5 units. The biggest change was 1.3 units in the blank control.

Validity criteria fulfilled:

yes

Conclusions:

The NOEC and EC50 values based on effects on growth rate were 56 and >100 (nominal) mg/L, respectively.

Executive summary:

A study was conducted to determine the effects of the test substance on algal growth using Desmodesmus subspicatus over an exposure period of 72 h, according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. A main test at concentrations of 10, 18, 32, 56 and 100 mg/L and blank control was performed. The test design included three replicates at each concentration and six replicates for the blank control. Analytical verification of test substance concentrations was conducted by DOC measurement. Due to low solubility of the test substance, there was a marginal difference of the DOC concentration in the control and treatment groups. Therefore, results were based on nominal concentrations. The cell concentration of each replicate was determined by measuring the absorption of the solutions at 440 nm every 24 h with a spectral photometer. On the basis of measured absorption values, cell numbers, growth rate, yield and inhibition were calculated. Significant inhibition of algal growth was observed at the highest concentration (100 mg/L). The 72 h ErC50 and EYC50 of the reference substance (potassium dichromate) were 1.1 and 0.39 mg/L, respectively, which were within the required range. All the validity criteria were met in this study. Under the study conditions, the NOEC, LOEC, EC10 and EC50 values based on the effects on yield were 56, 100, 12 and >100 mg/L (nominal), respectively. The NOEC, LOEC, EC10 and EC50 based on effects on growth rate were 56, 100, >100 and >100 mg/L (nominal), respectively (Muckle, 2015).

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

56 mg/L

Additional information

A study was conducted to determine the effects of the test substance on algal growth using Desmodesmus subspicatus over an exposure period of 72 h, according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. A main test at concentrations of 10, 18, 32, 56 and 100 mg/L and blank control was performed. The test design included three replicates at each concentration and six replicates for the blank control. Analytical verification of test substance concentrations was conducted by DOC measurement. Due to low solubility of the test substance, there was a marginal difference of the DOC concentration in the control and treatment groups. Therefore, results were based on nominal concentrations. The cell concentration of each replicate was determined by measuring the absorption of the solutions at 440 nm every 24 h with a spectral photometer. On the basis of measured absorption values, cell numbers, growth rate, yield and inhibition were calculated. Significant inhibition of algal growth was observed at the highest concentration (100 mg/L). The 72 h ErC50 and EYC50 of the reference substance (potassium dichromate) were 1.1 and 0.39 mg/L, respectively, which were within the required range. All the validity criteria were met in this study. Under the study conditions, the NOEC, LOEC, EC10 and EC50 values based on the effects on yield were 56, 100, 12 and >100 mg/L (nominal), respectively. The NOEC, LOEC, EC10 and EC50 based on effects on growth rate were 56, 100, >100 and >100 mg/L (nominal), respectively (Muckle, 2015).