reaction product of aminoacids and palmitoyl chloride

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:

The study was conducted between 11 November 2008 and 16 October 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2010

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

Principles of method if other than guideline:

LCE08089 is known to be poorly soluble in water. 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. Previous experience gained from studies conducted on poorly water soluble test materials has shown that a mixing period of 24 hours is 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.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid

Details on test material:

Sponsor's identification: LCE08089
Product code: LCE08044
Description: white solid
Batch number: 0901600021
Date received: 31 July 2009
Storage conditions: room temperature in the dark

Specific details on test material used for the study:

Sponsor's identification: LCE08089
Product code: LCE08044
Description: white solid
Batch number: 0901600021
Date received: 31 July 2009
Storage conditions: room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/l to confirm that no effect on algal growth was observed

Samples were taken from the uninoculated control and each loading rate WAF test group at 0 and 72 hours for this analysis

Duplicate samples were taken and stored frozen (approximately -20 degC) for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

Procedure

Range-finding test

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 Accommodated 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 100 ml 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 material (20 and 200 mg) were each separately added to the surface of 2 litres of culture medium to give the 10 and 100 mg/l loading rates respectively. 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 mixtures allowed to stand for 1 hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test material was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). 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. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 10 and 100 mg/l loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed there to be no micro-dispersions of test material present.

An aliquot (200 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (4.2 ml) to give the required test concentrations of 10 and 100 mg/l loading rate WAF.
The control group was maintained under identical conditions but not exposed to the test material.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1ºC 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.

After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

Definitive test

Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/l to confirm that no effect on algal growth was observed.

Experimental Preparation

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. Microscopic observations made on the WAF indicated that a significant amount of dispersed test material was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 cm in length). 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. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic observations of the WAF were performed after filtering and showed there to be no particles or micro-dispersions of test material present.
An aliquot (1 litre) of the WAF was inoculated with algal suspension (21.3 ml) to give the required test concentration of 100 mg/l loading rate WAF.
An additional 150 ml of the control and 100 mg/l loading rate WAF containing no algal cells was run alongside the test to provide samples for Total Organic Carbon (TOC) analysis at 0 and 72 hours (see Appendix 4). It was considered appropriate to omit the presence of algal cells in the samples prepared for TOC analysis as their presence would artificially increase the TOC concentration obtained.

Physico-chemical measurements
The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.

Vortex depth measurements
The vortex depth was recorded at the start and end of the mixing period - (see Table 5) - (in section any other information on materials and methods)

Evaluation of data
Comparison of growth rates and comparison of yield please see in attached section.

Determination of EL*x values
EL*x values were determined by inspection of the growth rate, yield and biomass integral data after 72 hours.

Statistical analysis
A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate, yield and biomass integral data after 72 hours for the control and the 100 mg/l loading rate 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).

Validation Criteria
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%.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test was carried out using Desmodesmus subspicatus strain CCAP 276/20. Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1deg C.

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 aninitial 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 deg C until the algal cell density was approximately 104 – 105 cells/ml.

A positive control (Harlan Laboratories Ltd Project Number: 0039/1088) used potassium dichromate as the reference material.

Details are given in section - results with reference substance (positive control)

The positive control was conducted between 26 May 2009 and 29 May 2009.

Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture. See below:
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 Na
OH or HCl.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

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:

The pH values of the control cultures (see Table 2) - (in section any other information on results) were observed to increase from pH 7.3 at 0 hours to pH 7.5 – 7.6 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Dissolved oxygen:

Not recorded.

Salinity:

freshwater used

Nominal and measured concentrations:

The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal loading rates of 10 and 100 mg/l
Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/l to confirm that no effect on algal growth was observed.

Details on test conditions:

Exposure conditions
As in the range-finding test 250 ml glass conical flasks were used. Six flasks each containing 100 ml of test preparation were used for the control and 100 mg/l loading rate WAF treatment group.
The control group was maintained under identical conditions but not exposed to the test material.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 1.88 x 105 cells per ml. Inoculation of 1 litre of test medium with 21.3 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.
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, 23, 50 and 72 hours and the cell densities 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:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL not stated

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL not stated

Details on results:

RESULTS
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 -see any other information on results section.
The results showed no effect on growth at 10 and 100 mg/l loading rate WAF.

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

Definitive Test
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2 (see any other information on results section). Daily specific growth rates for the control cultures are given in Table 3 (see any other information on results section). Growth rates, yield and biomass integral values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4 (see any other information on results section)


The mean cell densities versus time for the definitive test are presented in Figure 1-see attached.

Validation criteria
The following data show that the cell concentration of the control cultures increased by a factor of 99 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 : 4.42 x 103 cells per ml
Mean cell density of control at 72 hours : 4.38 x 105 cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 21% 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 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

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.
Accordingly the following results were determined from the data:

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 Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant differences (P0.05), 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.
5.2.2.2

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%.

Statistical analysis of the yield data was carried out as before. There were no statistically significant differences between the control and 100 mg/l loading rate WAF (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 100 mg/l loading rate WAF.

Inhibition of biomass integral
EbL*10 (0 - 72 h) : >100 mg/l loading rate WAF
EbL*20 (0 - 72 h) : >100 mg/l loading rate WAF
EbL*50 (0 - 72 h) : >100 mg/l loading rate WAF
where EbL*x is the loading rate that reduced biomass integral by x%.

Statistical analysis of the biomass integral data was carried out as before. There were no statistically significant differences (P<0.05), between the control and 100 mg/l loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on biomass integral was 100 mg/l loading rate WAF.

Physico-chemical measurements
The pH of each control and test flask are given in table 2 (see in any other information on results section)

Vortex depth measurements
The vortex depth was recorded at the start and end of the mixing period. (see table 5 in any other information on materials section).

Results with reference substance (positive control):

A positive control (Harlan Laboratories Ltd Project No: 0039/1088) 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) : 0.79 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:

Statistical analysis
A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate, yield and biomass integral data after 72 hours for the control and the 100 mg/l loading rate 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

Observationson 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 the start of stirring the 100 mg/l loading rate WAF was observed to have formed a clear colourless media column with test material floating at the media surface and suspended within the dimple. After stirring the WAF was observed to have formed a clear colourless media column with test material floating at the media surface and dispersed throughout the media column. Following the 1-Hour standing period the WAF was observed to have formed a clear colourless media column with test material floating at the media surface, suspended within the media column and settled on the bottom of the mixing vessel. 

Microscopic examination of the WAF indicated that micro-dispersions of test material were present and as such it was considered appropriate to remove the aqueous phase for testing by filtration through a glass wool plug. Following filtration microscopic examination of the WAF showed there to be no micro-dispersions of test material present.

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 test cultures were observed to be green dispersions.

 RESULTS

 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 - see below

The results showed no effect on growth 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.

Table 1              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/Biomass Integral
Control	R1	5.22E+03	2.61E+05
	R2	4.50E+03	3.10E+05	-	-
	Mean	4.86E+03	2.85E+05
10	R1	4.75E+03	3.61E+05
	R2	4.70E+03	2.85E+05	[4]	[14]
	Mean	4.72E+03	3.23E+05
100	R1	4.45E+03	3.23E+05
	R2	4.54E+03	2.64E+05	[2]	[3]
	Mean	4.49E+03	2.94E+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.

R₁and R₂= Replicates 1 and 2

DefinitiveTest

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2 - see below. 

Daily specific growth rates for the control cultures are given in Table 3 - see below. Growth rates, yield and biomass integral values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4 - see below.

The mean cell densities versus time for the definitive test are presented in Figure 1 - see attachment 1.

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	23 h	50 h	72 h	72 h
Control	R1	7.3	4.55E+03	1.40E+04	8.16E+04	4.16E+05	7.5
	R2	7.3	4.51E+03	1.39E+04	8.13E+04	4.62E+05	7.6
	R3	7.3	4.68E+03	1.28E+04	7.07E+04	3.64E+05	7.6
	R4	7.3	4.44E+03	1.81E+04	6.04E+04	4.20E+05	7.5
	R5	7.3	4.14E+03	1.75E+04	7.41E+04	4.23E+05	7.5
	R6	7.3	4.22E+03	1.63E+04	8.93E+04	5.41E+05	7.6
	Mean		4.42E+03	1.54E+04	7.62E+04	4.38E+05
100	R1	6.6	4.49E+03	1.50E+04	4.89E+04	4.86E+05	7.4
	R2	6.6	4.34E+03	1.51E+04	8.09E+04	4.66E+05	7.4
	R3	6.6	3.96E+03	1.40E+04	6.83E+04	4.61E+05	7.4
	R4	6.6	4.40E+03	1.18E+04	6.72E+04	4.40E+05	7.4
	R5	6.6	4.66E+03	1.27E+04	5.02E+04	4.50E+05	7.4
	R6	6.6	4.64E+03	1.72E+04	6.79E+04	4.49E+05	7.4
	Mean		4.42E+03	1.43E+04	6.39E+04	4.59E+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.

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	0.054	0.065	0.074
	R2	0.054	0.066	0.079
	R3	0.051	0.063	0.075
	R4	0.066	0.045	0.088
	R5	0.064	0.054	0.079
	R6	0.061	0.063	0.082
	Mean	0.058	0.059	0.080

R₁- R₆= Replicates 1 to 6

Table 4              Inhibition of Growth Rate, Yield and Biomass Integral in the Definitive Test

Nominal Loading Rate (mg/l)		Growth Rate (cells/ml/hour)		Yield (cells/ml)		Biomass Integral (cells/ml/hour)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*	0 – 72 h	% Inhibition
Control	R1	0.065		4.11E+05		6.68E+06
	R2	0.066		4.57E+05		7.18E+06
	R3	0.063		3.60E+05		5.82E+06
	R4	0.065	-	4.15E+05	-	6.30E+06	-
	R5	0.065		4.19E+05		6.66E+06
	R6	0.068		5.37E+05		8.31E+06
	Mean	0.065		4.33E+05		6.82E+06
	SD	0.002		5.97E+04		8.55E+05
100	R1	0.067	[3]	4.81E+05		6.68E+06	2
	R2	0.066	[2]	4.62E+05		7.24E+06	[6]
	R3	0.066	[2]	4.57E+05		6.85E+06	0
	R4	0.065	0	4.36E+05		6.54E+06	4
	R5	0.066	[2]	4.45E+05		6.25E+06	8
	R6	0.066	[2]	4.44E+05		6.79E+06	0
	Mean	0.066	[2]	4.54E+05	[5]	6.73E+06	1
	SD	0.001		1.62E+04		3.32E+05

*In accordance with the OECD test guideline only the mean value for yield 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 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.
* EL = Effective Loading Rate

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) 440/2008.

Methods.

Information provided by the Sponsor indicated that the test material was a mixture of components. Given this and also the poor solubility of the test material in water it was considered that the most suitable method of preparation for this material was as a Water Accommodated Fraction (WAF).

Method development work conducted for the determination of dissolved test material concentrations using HPLC-MS analysis gave a good response in single ion mode at 354 and 396 m/z. However, as the test material is a mixture of components it was considered that chemical analysis was not suitable for detecting the total dissolved test material concentration present. As such it was considered appropriate to determine the dissolved test material concentration present in the test samples by Total Organic Carbon (TOC) analysis.

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.

Total Organic Carbon (TOC) analysis of the test preparations at 0 hours showed a measured concentration of 6.01 mg C/l was obtained whilst a decline in measured concentrations was observed at 72 hours to 3.03 mg C/l. As TOC analysis is non-stability indicating this decline in measured concentrations was considered to be due to possible adsorption of the test material to the glassware and/or biological matter present.

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.

*EL= Effective Loading Rate

 CONCLUSION

The effect of the test material on the growth of Desmodesmus subspicatus 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