Eldew APS-307

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 12 February 2009 and 30 April 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

Report date:

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

Details on test material:

- Name of test material (as cited in study report): Eldew APS-307
- Substance type: pale yellow waxy solid block
- Physical state: solid
- Lot/batch No.: 809114
- Stability under test conditions: unstable, based on the preliminary stability analysis in culture medium.
- Storage condition of test material: room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
A C8 (EC) solid phase extraction (SPE) cartridge was sequentially pre-conditioned with methanol and water. A volume of test sample was eluted through the cartridge and the cartridge dried. The test material was eluted from the cartridge with methanol and made to volume to give a final theoretical concentration of approximately 10 mg/l.

Nominal concentration of 10 mg/L

- Sampling method:
Samples were taken from the control (replicates R1- R6pooled) and the 100 mg/l loading rate WAF test group (replicates R1- R3and R4- R6 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 0 hours and stored at approximately ‑20ºC for further analysis if necessary. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.
The method of analysis, stability, recovery and test preparation analyses are described in Appendix 4 (Attachment 2).


- Sample storage conditions before analysis: -20 °C.

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.

An amount of test material (250 mg) was added to the surface of 2.5 litres of culture medium to give the 100 mg/l loading rate. After the addition of the test material, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test material to be present.

An aliquot (2 litres) of the WAF was inoculated with algal suspension (13.3 ml) to give the required test concentration of 100 mg/l loading rate WAF.
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 72 hours.

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test material in cases where the test material is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test material with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test material and water phase. At the completion of mixing, the test material phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test material and/or leachates from the test material). Exposures are expressed in terms of the original concentration of test material in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test material in the WAF.

- Eluate:
Not applicable

- Controls:
A positive control (Harlan Laboratories Ltd Project No: 0039/1066) used potassium dichromate as the reference 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.

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

- Chemical name of vehicle :
Not applicable

- Concentration of vehicle in test medium:
Not applicable

- Evidence of undissolved material :
At both the start and end of the mixing period and following the I-Hour settlement period the WAF was observed to have formed a clear colourless media column with solid blobs of test material floating at the media surface.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name:
Green Algae
Master cultures were maintained in the laboratory by the periodic replenishment of culture medium.

- Strain:
Strain CCAP 276/20

- Source:
Obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland, UK.

- 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 10E03 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 deg C until the algal cell density was approximately 10E04 - 10E05 cells/mL.

- 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.

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.

- Any deformed or abnormal cells observed:
There were no abnormalities detected in any of the control or test cultures.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

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.

Test conditions

Hardness:

Not recorded.

Test temperature:

Temperature was maintained at 24 ± 1ºC throughout the test.

pH:

7.3 - 7.4 at 0 hours; 7.6 - 7.8 at 72 hours.

Dissolved oxygen:

Not recorded.

Salinity:

freshwater used

Nominal and measured concentrations:

Based on the result of the range-finding test a "limit test" was conducted at a single nominal loading rate of 100 mg/L was used.

Details on test conditions:

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

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

- Renewal rate of test solution (frequency/flow rate): No

- Initial cells density: cell density of approximately 4 x 10E+03 cells/ml.

- Control end cells density: 10E+04 – 10E+05 cells/mL.

- No. of vessels per concentration (replicates): 6

- No. of vessels per control (replicates): 6


GROWTH MEDIUM

For the purpose of the definitive test, the test material was dissolved directly in culture medium.


TEST MEDIUM
See appendix 2

OTHER TEST CONDITIONS
- Sterile test conditions: No

- Adjustment of pH:
No adjustments recorded

- Photoperiod:
Continous illumination

- Light intensity and quality:
7000 lux

EXPOSURE CONDITIONS :

Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 6.03 x 10E05 cells per ml. Inoculation of 2 litres of test medium with 13.3 ml of this algal suspension gave an initial nominal cell density of 4 x 10E03 cells per ml and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
Samples were taken at 0, 22, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

EFFECT PARAMETERS MEASURED : Algal growth
- 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
Based on the result of the range-finding test a "limit test" was conducted at a single nomonal loading rate of 100 mg/L.


- Range finding study:

Due to the low aqueous solubility and complex nature of the test material for the purposes of the test the test material was prepared as a water accomodated Fraction (WAF).

The loading rate to be, used in the definitive test was determined by a preliminary range finding test. The range finding test was conducted by exposing desmodesmus subspicatus cells to a series of nominal loading rates of 10 and 100 mg/l for a period of 72 hours.

The test was conducted in 250 ml glass conical flasks each containing 100ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.

Amounts of test materials (20 and 200mg) were each separately added to the surface of 2 litres of culture medium to give the 10 and 100mg/l loading rates respectively. After the addition of the test material, the culture medium was stirred by a magnetic stirrer using the stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5cm from the bottom of the vessel. Alength of tygon tubing was inserted into the glass tube and pushed through the nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100ml discarded) to give the 10 and 100mg/l loading rate WAFs Microscopic inspection of the WAFs showed no micro dispersions or undissolved test material to be present.

An aliquot (250ml) of each of the loading rate WAFs was separately innoculated with algal suspension (2.6ml) to give the required test concentrations of 10 and 100mg/l.

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

At the start of the rangefinding test a sample of each test and control culture was removed and the cell density determined using a coulter Multisizer Particle Counter.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loading rate WAF

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loading rate WAF

Basis for effect:

growth rate

Details on results:

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.

- Exponential growth in the control (for algal test): yes

Growth data
From the data given in Tables 2 and 4, it is clear that the growth rate (r), yield (y) and biomass (b) of Desmodesmus subspicatus (CCAP 276/20) were not affected by the presence of the test material over the 72-Hour exposure period.
It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Inhibition of growth rate

ErL*10(0 - 72 h)          : > 100 mg/l loading rate WAF
ErL*20(0 - 72 h)          : > 100 mg/l loading rate WAF
ErL*50(0 - 72 h)          : > 100 mg/l loading rate WAF

where ErL*x is the loading rate that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and 100 mg/l loading rate WAF test group using a Student’s t-test incorporating's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant decreases in growth rate (P³0.05), between the control and 100 mg/l loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 100 mg/l loading rate WAF.

*EL = Effective Loading rate
Inhibition of yield


EyL*10(0 - 72 h)         : > 100 mg/l loading rate WAF
EyL*20(0 - 72 h)         : > 100 mg/l loading rate WAF
EyL*50(0 - 72 h)         : > 100 mg/l loading rate WAF

where EyL*x is the loading rate that reduced yield by x%.

There were no statistically significant decreases in yield between the control and 100 mg/l loading rate WAF (P³0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 100 mg/l loading rate WAF.


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
Observations on the test media were carried out during the mixing and testing of the WAF.
At both the start and end of the mixing period and following the 1-Hour settlement period the WAF was observed to have formed a clear colourless media column with solid blobs of test material floating at the media surface.
At the start of the test all control and 100 mg/l loading rate WAF test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and 100 mg/l loading rate WAF test cultures were observed to be pale green dispersions.



- 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

Results with reference substance (positive control):

A positive control (Harlan Laboratories Ltd Project No: 0039/1066) used potassium dichromate as the reference 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) : 0.52 mg/l, 95% confidence limits 0.43 – 0.62 mg/l
EyC50 (0 – 72 h) : 0.29 mg/l, 95% confidence limits 0.25 – 0.33 mg/l
No Observed Effect Concentration (NOEC) based on growth rate : 0.125 mg/l
No Observed Effect Concentration (NOEC) based on yield : 0.125 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate : 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield : 0.25 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

 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 Table1.

The results showed no significant effect on growth rate at 10 and 100 mg/l loading rate WAF.

Based on this information a single loading rate of six replicates of 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no effect on growth was observed.

Observations on test material solubility

Observations on the test media were carried out during the mixing and testing of the WAF. At both the start and end of the mixing period and following the 1-Hour settlement period the WAF was observed to have formed a clear colourless media column with solid blobs of test material floating at the media surface. At the start of the test all control and 100 mg/l loading rate WAF test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and 100 mg/l loading rate WAF test cultures were observed to be pale green dispersions.

Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 29 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                      :   4.70 x 10³cells per ml
Mean cell density of control at 72 hours                   :   1.34 x 10⁵cells per ml

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

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 6% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Table1              Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Loading Rate (mg/l)		Cell Densities*(cells per ml)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	4.42E+03	2.88E+05
	R2	4.42E+03	3.13E+05	-	-
	Mean	4.42E+03	3.00E+05
10	R1	4.15E+03	2.81E+05
	R2	4.12E+03	2.86E+05	0	6
	Mean	4.14E+03	2.84E+05
100	R1	4.12E+03	1.86E+05
	R2	4.14E+03	1.90E+05	10	38
	Mean	4.13E+03	1.88E+05

 

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

R_{1 -}R₆= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

Table 2              Cell Densities and pH Values in the DefinitiveTest

Nominal Loading Rate (mg/l)		pH	Cell Densities*(cells per ml)				pH
		0 h	0 h	22h	48 h	72 h	72 h
Control	R1	7.4	4.27E+03	9.84E+03	4.29E+04	1.49E+05	7.8
	R2	7.4	4.26E+03	7.08E+03	2.82E+04	1.14E+05	7.8
	R3	7.3	4.78E+03	8.70E+03	2.77E+04	9.77E+04	7.7
	R4	7.3	5.28E+03	7.97E+03	3.15E+04	1.45E+05	7.6
	R5	7.3	5.64E+03	6.68E+03	2.56E+04	1.51E+05	7.6
	R6	7.3	3.95E+03	6.68E+03	3.02E+04	1.48E+05	7.6
	Mean		4.70E+03	7.82E+03	3.10E+04	1.34E+05
100	R1	7.2	4.10E+03	8.18E+03	4.09E+04	2.20E+05	7.6
	R2	7.2	3.78E+03	8.64E+03	3.54E+04	2.61E+05	7.6
	R3	7.2	4.20E+03	8.36E+03	3.58E+04	2.54E+05	7.6
	R4	7.2	5.05E+03	8.17E+03	3.52E+04	2.41E+05	7.6
	R5	7.2	4.40E+03	7.74E+03	3.58E+04	2.15E+05	7.6
	R6	7.2	5.58E+03	8.10E+03	2.98E+04	2.52E+05	7.6
	Mean		4.52E+03	8.20E+03	3.55E+04	2.41E+05

 

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

R_{1 -}R₆= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

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

			Daily Specific Growth Rate (cells/ml/hour)				Coefficient of variation (%)
			Day 0 - 1		Day 1 - 2			Day 2 - 3
Control		R1		0.041		0.057		0.052		16
		R2		0.026		0.053		0.058		37
		R3		0.035		0.045		0.052		20
		R4		0.031		0.053		0.064		35
		R5		0.023		0.052		0.074		52
		R6		0.023		0.058		0.066		47
		Mean		0.030		0.053		0.061		35

 

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

R_{1 -}R₆= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

Table 4              Inhibition of Growth Rate and Yieldin the Definitive Test

Nominal Loading Rate (mg/l)		Growth Rate (cells/ml/hour)		Yield (cells/ml)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.050		1.45E+05
	R2	0.046		1.09E+05
	R3	0.044		9.29E+04
	R4	0.050	-	1.40E+05	-
	R5	0.050		1.46E+05
	R6	0.050		1.44E+05
	Mean	0.048		1.29E+05
	SD	0.003		2.26E+04
100	R1	0.056	[17]	2.16E+05
	R2	0.058	[21]	2.58E+05
	R3	0.058	[21]	2.50E+05
	R4	0.057	[19]	2.36E+05
	R5	0.055	[15]	2.10E+05
	R6	0.058	[21]	2.46E+05
	Mean	0.057	[19]	2.36E+05	[82]
	SD	0.001		1.91E+04

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

R_{1 -}R₆= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

 

Table 5              Vortex Depth Measurements at the Start and End of the Mixing Period

	Nominal Loading Rate (mg/l)
	Control		100
	*	+	*	+
Height of Media Column (cm)	15	15	15	15
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present

 

* = Start of mixing period

+ = End of mixing period

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 and gave EL*50 values of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Executive summary:

Introduction.A study was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. The method 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 a Water Accommodated Fraction (WAF) of the test material, at a single nominal loading rate of 100 mg/l (six replicate flasks) 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 EL*₅₀values of greater than 100 mg/L loading rate WAF and correspondingly the No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Analysis of the test preparations at 0 hours showed measured test concentrations of 0.0069 and 0.010 mg/l were obtained (replicates R₁-R₃and R₄-R₆respectively) whilst concentrations of 0.033 and 0.026 mg/L (replicates R₁-R₃ and R₄-R₆respectively) were obtained at 72 hours. The apparent increase in measured test concentrations over the test period was considered to be due to a change in peak profile between 0 and 72 hours. The additional peaks observed at 72 hours were considered to be due to the presence of degradation products as the preliminary stability analyses conducted indicated that the test material was unstable in culture medium.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole the results were based on nominal loading rates only.

Conclusion. The effect of the test material on the growth of Desmodesmus subspicafus has been investigated and gave EL*₅₀values of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

*EL = Effective Loading Rate