Reaction mass of 2-(3-ethylphenyl)oxirane and 2,2’-(1,3-phenylene)dioxirane and 2,2'-(1,4-phenylene)dioxirane

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:

Between 8 June 2009 and 16 July 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 19/08/2008 Date of Signature: 04/03/2009

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane

IUPAC Name:

Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane

Details on test material:

- Name of test material (as cited in study report): Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane
- Substance type: Organic, Multiconstituent
- Physical state: clear extremely pale yellow liquid
- Analytical purity: 80-95%
- Lot/batch No.: 200901317-17
- Date received: 26 May 2009
- Stability under test conditions: Stable
- Storage condition of test material: approximately 4°C in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Definitive Test - 1.0, 3.2, 10, 32 and 100 mg/l.

- Sampling method:
Samples were taken from the control (replicates R1 - R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis.

- Sample storage conditions before analysis:
Duplicate samples were taken at 0 and 72 hours and stored at approximately 20ºC for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
For the purpose of the definitive test, the test material was dissolved directly in culture medium.

Amounts of test material (100 and 32 mg) were each separately dissolved in culture medium with the aid of ultrasonication for approximately 5 minutes and the volume adjusted to 1 litre to give 100 and 32 mg/l stock solutions respectively. A series of dilutions was made from these stock solutions to give further stock solutions of 10, 3.2 and 1.0 mg/l. An aliquot (500 ml) of each of the stock solutions was separately inoculated with 7.3 ml of algal suspension to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/l.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 72 hours.

- Eluate: Not applicable

- Controls:
A positive control used potassium dichromate as the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.

An aliquot (250 ml) of each of the 0.125, 0.25, 0.50, 1.0 and 2.0 mg/l stock solutions was separately mixed with algal suspension (250 ml) to give the required test concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.

The test material was dissolved directly in culture medium. An amount of test material (50 mg) was dissolved in culture medium and the volume adjusted to 500 ml to give a 100 mg/l stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/l. An aliquot (250 ml) of each of the stock solutions was separately inoculated with algal suspension (1.9 ml) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/l.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

The control group was maintained under identical conditions but not exposed to the test material.

- Chemical name of vehicle : Not applicable

- Concentration of vehicle in test medium: Not applicable

At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and 1.0 mg/l test cultures were observed to be pale green dispersions, the 3.2 and 10 mg/l test cultures were observed to be very pale green dispersions, the 32 mg/l test cultures were observed to be extremely pale green dispersions whilst the 100 mg/l test cultures were observed to be clear colourless solutions.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green Algae

- Strain: Strain CCAP 276/20

- Source:
Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.

- Age of inoculum : Not recorded

- Method of cultivation:

Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 104 - 105 cells/ml.

ACCLIMATION

- Acclimation period: Not recorded.

- Culturing media and conditions:
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture with the addition of 500 mg/l of sodium bicarbonate to counteract the increase in pH due to algal growth in an enclosed system (Herman et al 1990).

Culture medium:
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l

The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
For the purposes of the range-finding and definitive test, additional sodium bicarbonate (500 mg/l) was added to the prepared culture medium prior to use.


- Any deformed or abnormal cells observed: None recorded.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

Not recorded.

Test temperature:

Temperature was maintained at 24 ± 1ºC throughout the test. The temperature within the incubator was recorded daily.

pH:

7.2 - 7.7

Dissolved oxygen:

Not recorded.

Salinity:

freshwater used

Nominal and measured concentrations:

The range-finding test (nominal): 0.10, 1.0, 10 and 100 mg/l.
Definitive test (nominal): 1.0, 3.2, 10, 32 and 100 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass conical flasks
- Type: closed
- Material, size, headspace, fill volume: Glass, 250ml
- Aeration: No aeration

- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable

- Renewal rate of test solution (frequency/flow rate): Not applicable

- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 2.73 x 10E5 cells per ml. Inoculation of 500 ml of test medium with 7.3 ml of this algal suspension gave an initial nominal cell density of 4 x 10E3 cells per ml and had no significant dilution effect on the final test concentration.

- Control end cells density: algal cell density was approximately 2.73 10E5 cells/ml

- No. of organisms per vessel: initial cell density of approximately 4 x 10E3 cells/ml.

- No. of vessels per concentration (replicates): Three replicate flasks per concentration.

- No. of vessels per control (replicates): Six replicate flasks.


GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM

For the purpose of the definitive test, the test material was dissolved directly in culture medium.
Amounts of test material (100 and 32 mg) were each separately dissolved in culture medium with the aid of ultrasonication for approximately 5 minutes and the volume adjusted to 1 litre to give 100 and 32 mg/l stock solutions respectively. A series of dilutions was made from these stock solutions to give further stock solutions of 10, 3.2 and 1.0 mg/l. An aliquot (500 ml) of each of the stock solutions was separately inoculated with 7.3 ml of algal suspension to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/l.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 72 hours.



OTHER TEST CONDITIONS
- Sterile test conditions: Not stated in report

- Adjustment of pH:
No adjustments recorded

- Photoperiod:
Continous illumination

- Light intensity and quality:
warm white lighting (380 – 730 nm)


EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations:
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

- Chlorophyll measurement:
Not recorded

TEST CONCENTRATIONS
Definitive test: 1.0, 3.2, 10, 32 and 100 mg/l.

Range finding study: 0.10, 1.0, 10 and 100 mg/l

VALIDATION:
The results of the test are considered valid if the following performance criteria are met:
* The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.

* The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.

* The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% Confidence limits of 5.6 - 7.8

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits of 16 - 26

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits 16 - 26 mg/l

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% confidence limits 5.6 - 7.8 mg/l

Effect concentrations 5

Duration:

72

Dose descriptor:

LOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 95% Confidence limits of 5.6 - 7.8

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence limits of 5.6 - 7.8

Details on results:

Observations on cultures
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Observations on test material solubility
At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and 1.0 mg/l test cultures were observed to be pale green dispersions, the 3.2 and 10 mg/l test cultures were observed to be very pale green dispersions, the 32 mg/l test cultures were observed to be extremely pale green dispersions whilst the 100 mg/l test cultures were observed to be clear colourless solutions.

- Any stimulation of growth found in any treatment: None

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None recorded

Range-finding Test
The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test material during the range-finding test are given in Table 1.
The results showed no effect on growth at the test concentrations of 0.10 and 1.0 mg/l. However, growth was observed to be reduced at 10 and 100 mg/l.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/l were selected for the definitive tes

Growth data
From the data given in Tables 2 and 4, it is clear that the growth rate (r) and yield (y) ofDesmodesmus subspicatus(CCAP 276/20) were affected by the presence of the test material over the 72-Hour exposure period.

Accordingly the following results were determined from the data:

Inhibition of growth rate
ErC10(0 - 72 h)           : 4.3 mg/l
ErC20(0 - 72 h)           : 7.7 mg/l
ErC50(0 - 72 h)           : 21 mg/l; 95% confidence limits 16 - 26 mg/l
where ErCxis the test concentration that reduced growth rate by x%.

There were no statistically significant differences between the control, 1.0 and 3.2 mg/l test concentrations (P³0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 3.2 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 10 mg/l.
 
Inhibition of yield
EyC10(0 - 72 h)          : 3.0 mg/l
EyC20(0 - 72 h)          : 4.0 mg/l
EyC50(0 - 72 h)          : 6.6 mg/l; 95% confidence limits 5.6 – 7.8 mg/l
where EyCxis the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out as described above. There were no statistically significant differences between the control and 1.0 mg/l test concentration (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 1.0 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 3.2 mg/l.


 

Results with reference substance (positive control):

Potassium dichromate was used as the positive control material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference material gave the following results:

ErC50 (0 – 72 h) : 0.79 mg/l
EyC50 (0 – 72 h) : 0.30 mg/l, 95% confidence limits 0.27 – 0.34 mg/l

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/l
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/l

The results from the positive control with potassium dichromate were within the normal ranges for this reference material.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate, yield and biomass integral data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Any other information on results incl. tables

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 53 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours                      :   5.28 x 10³cells per ml
Mean cell density of control at 72 hours                   :   2.81 x 10⁵cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 31% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

Table 1 Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentration (mg/l)		Cell Densities*(cells per ml)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	4.10E+03	4.75E+05
	R2	4.03E+03	3.44E+05	-	-
	Mean	4.06E+03	4.10E+05
0.10	R1	4.03E+03	3.70E+05
	R2	4.00E+03	4.14E+05	0	4
	Mean	4.02E+03	3.92E+05
1.0	R1	4.06E+03	4.06E+05
	R2	4.08E+03	4.81E+05	[2]	[8]
	Mean	4.07E+03	4.43E+05
10	R1	4.10E+03	1.70E+05
	R2	3.98E+03	1.78E+05	19	58
	Mean	4.04E+03	1.74E+05
100	R1	4.14E+03	8.62E+03
	R2	4.08E+03	5.47E+03	89	99
	Mean	4.11E+03	7.04E+03

*Cell densities represent thean number of cells per ml calculated from thean of the cell counts from 3 counts for each of the replicate flasks.

R₁and R₂= Replicates 1 and 2

Table 2. Cell Densities and pH Values in the Definitive Test

Nominal Concentration (mg/l)		pH	Cell Densities*(cells per ml)				pH
		0 h	0 h	26 h	48 h	72 h	72 h
Control	R1	7.2	5.29E+03	1.53E+04	4.35E+04	2.62E+05	7.6
	R2	7.2	5.18E+03	1.91E+04	4.25E+04	2.71E+05	7.7
	R3	7.2	5.23E+03	1.42E+04	4.15E+04	2.82E+05	7.7
	R4	7.2	5.72E+03	1.58E+04	4.30E+04	2.84E+05	7.7
	R5	7.2	5.20E+03	1.55E+04	4.19E+04	3.03E+05	7.7
	R6	7.2	5.06E+03	1.43E+04	4.23E+04	2.84E+05	7.7
	Mean		5.28E+03	1.57E+04	4.25E+04	2.81E+05
1.0	R1	7.2	4.80E+03	1.70E+04	4.27E+04	2.81E+05	7.7
	R2	7.2	4.96E+03	1.49E+04	4.11E+04	2.84E+05	7.6
	R3	7.2	4.89E+03	1.90E+04	4.11E+04	2.86E+05	7.6
	Mean		4.88E+03	1.70E+04	4.16E+04	2.84E+05
3.2	R1	7.1	5.07E+03	1.31E+04	3.47E+04	2.72E+05	7.4
	R2	7.1	4.88E+03	1.42E+04	3.85E+04	2.40E+05	7.4
	R3	7.1	4.87E+03	1.97E+04	2.58E+04	2.39E+05	7.4
	Mean		4.94E+03	1.56E+04	3.30E+04	2.50E+05
10	R1	7.1	4.90E+03	1.33E+04	1.89E+04	6.48E+04	7.4
	R2	7.1	4.62E+03	1.43E+04	2.46E+04	6.21E+04	7.4
	R3	7.1	4.96E+03	1.30E+04	2.00E+04	7.93E+04	7.4
	Mean		4.83E+03	1.35E+04	2.12E+04	6.87E+04
32	R1	7.1	5.18E+03	4.62E+03	9.90E+03	2.74E+04	7.3
	R2	7.1	5.04E+03	6.77E+03	5.81E+03	2.24E+04	7.3
	R3	7.1	5.72E+03	5.07E+03	2.26E+03	2.40E+04	7.3
	Mean		5.31E+03	5.49E+03	5.99E+03	2.46E+04
100	R1	7.1	4.83E+03	4.05E+03	2.57E+03	4.50E+03	7.2
	R2	7.1	4.86E+03	4.22E+03	2.41E+03	4.94E+03	7.2
	R3	7.1	5.24E+03	5.98E+03	2.08E+03	3.12E+03	7.2
	Mean		4.98E+03	4.75E+03	2.35E+03	4.19E+03

*Cell densities represent thean number of cells per ml calculated from thean of the cell counts from 3 counts for each of the replicate flasks.

R₁- R₆= Replicates 1 to 6

Table 3. Daily Specific Growth Rates for the Control Cultures in the Definitive Test

		Daily Specific Growth Rate (cells/ml/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.052	0.047	0.075
	R2	0.060	0.036	0.077
	R3	0.049	0.049	0.080
	R4	0.053	0.046	0.079
	R5	0.052	0.045	0.082
	R6	0.049	0.049	0.079
	Mean	0.053	0.045	0.079

R₁- R₆= Replicates 1 to 6

Table 4. Inhibition Growth Rate and Yield in the Definitive Test

Nominal Concentration (mg/l)		Growth Rate (cells/ml/hour)		Yield (cells/ml)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.058		2.56E+05
	R2	0.059		2.66E+05
	R3	0.059		2.77E+05
	R4	0.059	-	2.78E+05	-
	R5	0.060		2.98E+05
	R6	0.059		2.79E+05
	Mean	0.059		2.76E+05
	SD	0.001		1.40E+04
1.0	R1	0.059	0	2.76E+05
	R2	0.059	0	2.80E+05
	R3	0.059	0	2.81E+05
	Mean	0.059	0	2.79E+05	[1]
	SD	0.000		2.34E+03
3.2	R1	0.059	0	2.67E+05
	R2	0.057	3	2.35E+05
	R3	0.057	3	2.34E+05
	Mean	0.058	2	2.45E+05	11
	SD	0.001		1.88E+04
10	R1	0.039	34	5.99E+04
	R2	0.038	36	5.75E+04
	R3	0.041	31	7.43E+04
	Mean	0.039	34	6.39E+04	77
	SD	0.002		9.09E+03
32	R1	0.027	54	2.22E+04
	R2	0.024	59	1.74E+04
	R3	0.025	58	1.83E+04
	Mean	0.025	57	1.93E+04	93
	SD	0.002		2.57E+03
100	R1	0.002	97	-3.36E+02
	R2	0.003	95	8.00E+01
	R3	-0.003	105	-2.12E+03
	Mean	0.001	99	-7.92E+02	100
	SD	0.003		1.17E+03

*In accordance with the OECD test guideline only thean value for yield for each test concentration is calculated

R₁– R₆= Replicates 1 to 6

SD = Standard Deviation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave the results in the table below.

Executive summary:

Introduction. A study was perford to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. Thethod followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 440/2008.

Methods. Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to an aqueous solution of the test material at concentrations of 1.0, 3.2, 10, 32 and 100 mg/l (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

Results. Exposure of Desmodesmus subspicatus to the test material gave the following results:

Response Variable	EC50(mg/l)	95% Confidence Limits (mg/l)			No Observed Effect Concentration (NOEC) (mg/l)	Lowest Observed Effect Concentration (LOEC) (mg/l)
Growth Rate	21	16	-	26	3.2	10
Yield	6.6	5.6	-	7.8	1.0	3.2

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations range from 85% to 111% of nominal and so the results are based on nominal test concentrations only.

Conclusion. The effect of the test material on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave the results in the table above.