Glutens, hydrolyzates, reaction products with lauroyl chloride, sodium salts

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:

between 14 November 2008 and 28 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.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Sponsor's identification: LCE08082
Description: extremely pale yellow liquid
Date received: 5 September 2008
Storage conditions: room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Verification of test concentrations
Information provided by the Sponsor indicated that the test material was a complex mixture of amino acids. Pre-study solubility work conducted indicated that the test material was readily water soluble and as such it was considered that the most suitable method of preparation for this material was as a direct solution in culture medium. Method development work conducted for the determination of dissolved test material concentrations using HPLC-MS analysis gave a good response in single ion mode with m/z 511.4 (see attached Appendix 4). However, as the test material is a mixture of various amino acid reaction products 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.


Total organic carbon analysis
In order to determine the carbon content of the test material and hence the dissolved test material concentration present in the test samples stock solutions were prepared, in triplicate, in deionised reverse osmosis water at a concentration of 500 mg/l. A sample (50 ml) of each of the triplicate stock solutions was then taken for TOC analysis and the mean of the three values calculated to give a mean test material carbon concentration. Samples were also taken for TOC analysis from one of the 500 mg/l preparations following filtration through a 0.2 μm Gelman Acrocap filter (first approximate 100 ml discarded in order to pre-condition the filter) and also following centrifugation at 40000 g for 30 minutes to determine whether the test material was completely in solution (see attached Appendix 3).
Analysis of the test preparations was carried out by TOC analysis. Samples were taken from the additional control and 100 mg/l test sample containing no algal cells at 0 and 72 hours for this analysis (see Appendix 3). Duplicate samples were taken and stored frozen (approximately -20 degC) for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Range-finding test
The test concentration 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 test concentrations of 0.10, 1.0, 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 used for each control and test concentration. The test material was dissolved directly in culture medium.
An amount of test material (100 mg) was dissolved in culture medium and the volume adjusted to 500 ml to give a 200 mg/l stock solution from which a series of dilutions was made to give further stock solutions of 20, 2.0 and 0.20 mg/l. An aliquot (100 ml) of each of the stock solutions was separately mixed with algal suspension (100 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.
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 concentration of 100 mg/l to confirm that at the maximum concentration given in the OECD/EEC Test Guidelines no effect on algal growth was observed.

Experimental Preparation
For the purpose of the definitive test, the test material was dissolved directly in culture medium.
An amount of test material (200 mg) was dissolved in culture medium and the volume adjusted to 2 litres to give a 100 mg/l stock solution. An aliquot (1 litre) of this stock solution was inoculated with algal suspension (24 ml) to give the required test concentration of 100 mg/l.
The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.
An additional 100 ml of the control and 100 mg/l test concentration containing no algal cells was run alongside the test to provide samples for Total Organic Carbon (TOC) analysis at 0 and 72 hours (see attached Appendix 3). 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.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Test Species
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 (Section 3.2). The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1°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 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 10E4 - 10E5 cells/ml.
A positive control (Harlan Laboratories Ltd Project Number: 0039/1088) used potassium dichromate as the reference material. Details of the positive control are given in Appendix 1. 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.
The culture medium is defined in attached Appendix 2.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

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 each test and control flask are given in Table 2.
The pH values of the control cultures (see Table 2) were observed to increase from pH 7.3 at 0 hours to pH 7.9 - 8.0 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:

TOC analysis of the control and 100 mg/L test preparation containing no algal cells was conducted at 0 and 72 hours. Analysis of the 100 mg/L test preparation at 0 hours showed a measured concentration of 96 mg/L was obtained. A decline in measured concentrations was observed at 72 hours to 27 mg/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 toxicity cannot be attributed to a signle component or mixture of components but to the test material as a whole the results were based on nominal test concentrations only.

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 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 1.61 x 10e5 cells per ml. Inoculation of 1 litre of test medium with 24 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, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Reference substance (positive control):

yes

Remarks:

potassium dichromate please see Appendix 1 Positive Control for full details.

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.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on 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.
The results showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and 100 mg/l.
Based on this information a single test concentration of six replicates, of 100 mg/l was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the maximum test concentration given in the OECD/EEC Test Guidelines no effect on growth was observed.
Definitive Test
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, yield and biomass integral values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.
The mean cell densities versus time for the definitive test are presented in attached Figure 1.
Validation criteria
The following data show that the cell concentration of the control cultures increased by a factor of 231 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 : 4.22 x 10E3 cells per ml
Mean cell density of control at 72 hours : 9.75 x 10E5 cells per ml
The mean coefficient of variation for section by section specific growth rate for the control cultures was 22% 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 1% 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 integral (b) of Desmodesmus subspicatus (CCAP 276/20) were not affected by the presence of the test material at a nominal test concentration of 100 mg/l over the 72-Hour exposure period.
It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l.
Accordingly the following results were determined from the data based on nominal test concentrations:
Inhibition of growth rate
ErC10 (0 - 72 h) : >100 mg/l
ErC20 (0 - 72 h) : >100 mg/l
ErC50 (0 - 72 h) : >100 mg/l
where ErCx is the test concentration that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and 100 mg/l 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 test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100 mg/l.
Inhibition of yield
EyC10 (0 - 72 h) : >100 mg/l
EyC20 (0 - 72 h) : >100 mg/l
EyC50 (0 - 72 h) : >100 mg/l
where EyCx is the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out as in Section 5.2.2.1. There were no statistically significant differences (P0.05), between the control and 100 mg/l test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 100 mg/l.
Inhibition of biomass integral
EbC10 (0 - 72 h) : >100 mg/l
EbC20 (0 - 72 h) : >100 mg/l
EbC50 (0 - 72 h) : >100 mg/l
where EbCx is the test concentration that reduced biomass integral (area under the growth curve) by x%.
Statistical analysis of the biomass integral data was carried out as in Section "Inhibition of growth rate". There were no statistically significant differences (p≥0.05), between the control and 100 mg/l test group and therefore the "No Observed Effect Concentration" (NOEC) based on biomass integral was 100 mg/l.
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 test cultures were observed to be green dispersions.
Physico-chemical measurements
The pH values of each test and control flask are given in Table 2. Temperature was maintained at 24 ± 1ºC throughout the test.
The pH values of the control cultures (see Table 2) were observed to increase from pH 7.3 at 0 hours to pH 7.9 - 8.0 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.
Total organic carbon analysis
Total Organic Carbon (TOC) analysis of the triplicate 500 mg/l stock solutions showed the test material contained approximately 12.41% carbon (see Appendix 3). The results from both the filtered and centrifuged test samples gave similar results to those analysed without any pre-treatment and as such it was considered that the test material was fully dissolved.
TOC analysis of the control and 100 mg/l test preparation containing no algal cells was conducted at 0 and 72 hours (see attached Appendix 3). Analysis of the 100 mg/l test preparation at 0 hours showed a measured concentration of 96 mg/l was obtained. A decline in measured concentrations was observed at 72 hours to 27 mg/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 toxicity cannot be attributed to a single component or mixture of components but to the test material as a whole the results were based on nominal test concentrations only.

Results with reference substance (positive control):

Appendix 1 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 and the 100 mg/l test concentration 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).

Table1              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/Biomass Integral
Control	R1	4.17E+03	1.80E+05
	R2	4.13E+03	2.23E+05	-	-
	Mean	4.15E+03	2.01E+05
0.10	R1	4.18E+03	2.19E+05
	R2	4.10E+03	2.41E+05	[4]	[15]
	Mean	4.14E+03	2.30E+05
1.0	R1	4.20E+03	2.98E+05
	R2	4.34E+03	2.59E+05	[7]	[39]
	Mean	4.27E+03	2.79E+05
10	R1	4.14E+03	2.95E+05
	R2	4.02E+03	2.31E+05	[7]	[31]
	Mean	4.08E+03	2.63E+05
100	R1	4.21E+03	2.85E+05
	R2	4.18E+03	3.00E+05	[9]	[46]
	Mean	4.19E+03	2.93E+05

*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

[Increase in growth compared to controls]

Table 2              Cell Densities and pH Values in the DefinitiveTest

Nominal Concentration (mg/l)		pH	Cell Densities*(cells per ml)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	7.3	3.65E+03	1.94E+04	1.45E+05	9.19E+05	8.0
	R2	7.3	4.20E+03	1.48E+04	1.58E+05	9.37E+05	8.0
	R3	7.3	3.94E+03	1.45E+04	1.70E+05	1.04E+06	8.0
	R4	7.3	4.56E+03	1.73E+04	1.54E+05	9.87E+05	7.9
	R5	7.3	4.77E+03	2.04E+04	1.58E+05	9.89E+05	7.9
	R6	7.3	4.21E+03	1.61E+04	1.57E+05	9.77E+05	7.9
	Mean		4.22E+03	1.71E+04	1.57E+05	9.75E+05
100	R1	7.1	3.91E+03	2.47E+04	1.45E+05	1.07E+06	7.8
	R2	7.1	4.22E+03	2.80E+04	1.40E+05	9.84E+05	7.8
	R3	7.1	3.70E+03	2.71E+04	1.37E+05	9.77E+05	7.7
	R4	7.1	4.44E+03	2.07E+04	1.56E+05	9.87E+05	7.7
	R5	7.1	4.04E+03	2.52E+04	1.41E+05	9.85E+05	7.7
	R6	7.1	3.77E+03	2.60E+04	1.53E+05	9.93E+05	7.7
	Mean		4.01E+03	2.53E+04	1.45E+05	9.99E+05

*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	0.066	0.084	0.077
	R2	0.055	0.099	0.074
	R3	0.054	0.102	0.076
	R4	0.061	0.091	0.077
	R5	0.068	0.085	0.076
	R6	0.058	0.095	0.076
	Mean	0.060	0.093	0.076

R₁- R₆= Replicates 1 to 6

Appendix 2      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*and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

*Elga Optima 15+ or Elga Purelab Option R-15 BP

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated and based on nominal test concentrations gave EC50 values of greater than 100 mg/l. The No Observed Effect Concentration was determined to be 100 mg/l.

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. 

Information provided by the Sponsor indicated that the test material was a complex mixture of amino acids. Pre-study solubility work conducted indicated that the test material was readily water soluble and as such it was considered that the most suitable method of preparation for this material was as a direct solution in culture medium. Method development work conducted for the determination of dissolved test material concentrations using HPLC-MS analysis gave a good response in single ion mode with m/z 511.4. However, as the test material is a mixture of various amino acid reaction products 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 an aqueous solution of the test material at a concentration 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 EC₅₀values of greater than 100 mg/l. The No Observed Effect Concentration was determined to be 100 mg/l.

Total Organic Carbon (TOC) analysis of the 100 mg/l test preparation at 0 hours showed a measured concentration of 96 mg/l was obtained. A decline in measured concentrations was observed at 72 hours to 27 mg/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 test concentrations only.

Conclusion.

 

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated and based on nominal test concentrations gave EC₅₀values of greater than 100 mg/l. The No Observed Effect Concentration was determined to be 100 mg/l.

Description of key information

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated and based on nominal test concentrations gave EC50 -72h values of greater than 100 mg/l.  The No Observed Effect Concentration was determined to be 100 mg/l.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L