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REACH

Chromium, 1-[2-[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]diazenyl]-2-naphthalenol 1-[2-[2-hydroxy-4(or 5)-nitrophenyl]diazenyl]-2-naphthalenol ammonium sodium complexes

EC number: 700-174-4 | CAS number: 1029600-34-7

Life Cycle description
Uses advised against

Ecotoxicological information

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:: 16 January 2009 and 6 February 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 guidelineopen all close all

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

Test material

Test material information

Details on test material:: - Name of test material (as cited in study report): VCBK3810
- Substance type: black powder
- Physical state: solid
- Lot/batch No.: N006746
- Stability under test conditions: stable
- Storage condition of test material: room temperature in the dark

Sampling and analysis

Analytical monitoring:: yes
Details on sampling:: - Concentrations:
nominal test concentrations of 0.0027 and 0.027 mg/l for a period of 72 hours.

- Sampling method:
A C18-E solid phase extraction (SPE) cartridge (packed with glass wool) was sequentially pre-conditioned with methanol and water. A volume of test sample acidified with orthophosphoric acid (4 drops/100 ml) was eluted through the cartridge and the cartridge dried. The test material was then eluted from the cartridge with mobile phase.

- Sample storage conditions before analysis:
Duplicate samples were taken and stored frozen (approximately -20 degC) for further analysis if necessary.

Test solutions

Vehicle:: no
Details on test solutions:: PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
Based on the results obtained from the media preparation trials conducted it was considered that the most appropriate method of preparation of the test material was the solvent spike method followed by removal of any undissolved test material by filtration to give a test concentration of 0.027 mg/l.
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 0.0027 and 0.027 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 prepared using a preliminary solution in methanol.
An amount of test material (200 mg) was dissolved in methanol and the volume adjusted to 10 ml to give a 200 mg/10 ml solvent stock solution. An aliquot (100 µl) of this 200 mg/10 ml solvent stock solution was dispersed in 1 litre of culture medium with the aid of magnetic stirring for approximately 10 minutes to give the required test concentration of 2.0 mg/l. Following stirring any undissolved test material was removed by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 500 ml discarded) to give a 0.027 mg/l stock solution from which a dilution was made to give a further stock solution of 0.0027 mg/l. An aliquot (200 ml) of each of the stock solutions was separately inoculated with algal suspension (3.2 ml) to give the required nominal test concentrations of 0.0027 and 0.027 mg/l.
The control and the solvent control groups were maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 µl/l of methanol.
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.

- 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.

- Chemical name of vehicle :
Not applicable

- Concentration of vehicle in test medium:
Not applicable

- Evidence of undissolved material :
At the start of the test all control and solvent control cultures were observed to be clear colourless solutions whilst the 0.42 mg/l test cultures were observed to be pale purple solutions. After the 72-Hour test period the control cultures were observed to be green dispersions, the solvent control cultures were observed to be pale green dispersions and the 0.42 mg/l test cultures were observed to be pale green/grey dispersions.

Test organisms

Test organisms (species):: Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:: TEST ORGANISM
- Common name:
Green Algae

- 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

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

- Any deformed or abnormal cells observed:
None recorded.

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 were observed to increase from pH 7.3 – 7.4 at 0 hours to pH 8.0 – 8.2 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.

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.
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 nominal concentration of 0.027 mg/l to confirm that at highest attainable concentration of 0.027 mg/l, no effect on algal growth was observed.
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 3.65 x 10E5 cells per ml. Inoculation of 1 litre of test medium with 11 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 6.24 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
For the purpose of the definitive test, the test material was dissolved directly in culture medium. . A standard solution was accurately prepared by dissolving the test material in methanol. An accurate volume of the standard solution was added to a known volume of test medium to achieve the required concentration of test material.

TEST MEDIUM

An amount of test material (200 mg) was dissolved in methanol and the volume adjusted to 10 ml to give a 200 mg/10 ml solvent stock solution. An aliquot (1000 µl) of this 200 mg/10 ml solvent stock solution was dispersed in 10 litres of culture medium with the aid of magnetic stirring for approximately 10 minutes to give the required test concentration of 2.0 mg/l prior to taking samples for chemical analysis after the following pre-treatments:

Untreated
Centrifugation at 10000 g for 30 minutes
Centrifugation at 40000 g for 30 minutes
Filtration through a 0.2 µm Gelman Acrocap filter (approximately 100 ml discarded in order to pre-condition the filter)
Filtration through a 0.2 µm Gelman Acrocap filter (approximately 500 ml discarded in order to pre-condition the filter)

The remainder of the 2.0 mg/l test concentration was returned to the magnetic stirrer and stirred for a further 48 hours with samples being taken for analysis after both 24 and 48 hours stirring.

OTHER TEST CONDITIONS
- Sterile test conditions: No data

- Adjustment of pH:
No adjustments recorded

- Photoperiod:
Not applicable

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

EXPOSURE CONDITIONS :

An amount of test material (200 mg) was dissolved in methanol and the volume adjusted to 10 ml to give a 200 mg/10 ml solvent stock solution. An aliquot (100 µl) of this 200 mg/10 ml solvent stock solution was dispersed in 1 litre of culture medium with the aid of magnetic stirring for approximately 10 minutes to give the required test concentration of 2.0 mg/l. Following stirring any undissolved test material was removed by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 500 ml discarded) to give a 0.027 mg/l stock solution from which a dilution was made to give a further stock solution of 0.0027 mg/l. An aliquot (200 ml) of each of the stock solutions was separately inoculated with algal suspension (3.2 ml) to give the required nominal test concentrations of 0.0027 and 0.027 mg/l.
The control and the solvent control groups were maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 µl/l of methanol.
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.

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.
Determination of ECx values
For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation using the Xlfit software package (IDBS). ECxvalues were then determined from the equation for the fitted line.

Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

- Chlorophyll measurement:
Not recorded

TEST CONCENTRATIONS
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 10, 20, 40, 80 and 160 mg/l.

- Range finding study:
The loading rates to be used in the definitive test were 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 completely filled 250 ml glass conical flasks sealed with ground glass stoppers to reduce evaporation. Two replicate flasks were prepared for each control and test concentration. The test material was prepared as a Water Accommodated Fraction (WAF).
Amounts of test material (24 and 230 mg) were each separately added to the surface of 2.4 and 2.3 litres of culture medium respectively in sealed vessels with minimal headspace 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. 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 10 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test material to be present.
An aliquot (1 litre) of each of the loading rate WAFs was separately inoculated with algal suspension (15.7 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.

POSITIVE CONTROL:
A positive control (Safepharm Laboratories Project Number: 0039/0994) used potassium dichromate as the reference material. An amount of reference material (100 mg) was dissolved in culture medium and the volume adjusted to 1 litre to give a 100 mg/l stock solution from which a series of dilutions was made to give further stock solutions of 10, 2.0, 1.0, 0.50, 0.25 and 0.125 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 was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Six replicate flasks were prepared for the control and three replicate flasks prepared for each test concentration.
The flasks were 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.

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 concentrationsopen all close all

Effect concentrations 1

Duration:: 0 h
Dose descriptor:: NOEC
Effect conc.:: 0.42 mg/L
Nominal / measured:: nominal
Conc. based on:: test mat.
Basis for effect:: other: growth rate
Remarks on result:: other: 95% CL not stated

Effect concentrations 2

Duration:: 0 h
Dose descriptor:: EC50
Effect conc.:: > 0.42 mg/L
Nominal / measured:: nominal
Conc. based on:: test mat.
Basis for effect:: growth rate
Remarks on result:: other: It was not possible to calculate 95% confidence limits for the ErL50 value as the data generated did not fit the models available for the calculation of confidence limits.

Effect concentrations 3

Duration:: 72 h
Dose descriptor:: EC50
Effect conc.:: > 0.32 mg/L
Nominal / measured:: meas. (geom. mean)
Conc. based on:: test mat.
Basis for effect:: growth rate
Remarks on result:: other: 95% CL not stated

Effect concentrations 4

Duration:: 72 h
Dose descriptor:: NOEC
Effect conc.:: 0.32 mg/L
Nominal / measured:: meas. (geom. mean)
Conc. based on:: test mat.
Basis for effect:: growth rate
Remarks on result:: other: 95% CL

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

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

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

Observations on test material solubility
At the start of the test all control and solvent control cultures were observed to be clear colourless solutions whilst the 0.42 mg/l test cultures were observed to be pale purple solutions. After the 72-Hour test period the control cultures were observed to be green dispersions, the solvent control cultures were observed to be pale green dispersions and the 0.42 mg/l test cultures were observed to be pale green/grey 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

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 0.0027 and 0.027 mg/l.
Based on this information a single nominal test concentration of six replicates, of 0.027 mg/l was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the highest attainable test concentration of 0.027 mg/l no effect on growth was observed.
Chemical analysis of the test preparations at 0 hours showed a mean measured concentration of 0.42 mg/l was obtained. This value was considerably higher than the predicted value obtained from the pre-study media preparation trial conducted. Further inspection of the pre-study media preparation trial results would suggest that an error occurred in the preparation of the solvent spike sample which in turn resulted in the comparably low value obtained from the centrifuged test sample.

Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 86 after 72 hours and the cell concentration of the solvent control cultures increased by a factor of 84 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 : 3.80 x 103 cells per ml
Mean cell density of control at 72 hours : 3.25 x 105 cells per ml
Mean cell density of solvent control at 0 hours : 3.61 x 103 cells per ml
Mean cell density of solvent control at 72 hours : 3.02 x 105 cells per ml
The mean coefficients of variation for section by section specific growth rate for the control and solvent control cultures were 32% and 34% respectively and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficients of variation for average specific growth rate for the control and solvent control cultures over the test period (0 – 72 h) were 5% and 2% respectively 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 0-Hour mean measured test concentration of 0.42 mg/l over the 72-Hour exposure period.
The test concentration of 0.42 mg/l was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the study under the OECD Guideline.
Accordingly the following results were determined from the data based on the 0-Hour mean measured test concentration:

Inhibition of growth rate
ErC10 (0 - 72 h) : > 0.42 mg/l
ErC20 (0 - 72 h) : > 0.42 mg/l
ErC50 (0 - 72 h) : > 0.42 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 solvent control and 0.42 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 solvent control and 0.42 mg/l test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.42 mg/l.

Inhibition of yield
EyC10 (0 - 72 h) : > 0.42 mg/l
EyC20 (0 - 72 h) : > 0.42 mg/l
EyC50 (0 - 72 h) : > 0.42 mg/l
where EyCx is the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out. There were no statistically significant differences (P0.05), between the solvent control and 0.42 mg/l test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.42 mg/l.

Inhibition of biomass integral
EbC10 (0 - 72 h) : > 0.42 mg/l
EbC20 (0 - 72 h) : > 0.42 mg/l
EbC50 (0 - 72 h) : > 0.42 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. There were no statistically significant differences (P0.05), between the solvent control and 0.42 mg/l test group and therefore the "No Observed Effect Concentration" (NOEC) based on biomass integral was 0.42 mg/l.
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
EbC50 (0 – 72 h) : 0.30 mg/l, 95% confidence limits 0.26 – 0.34 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
No Observed Effect Concentration (NOEC) based on biomass integral: 0.0625 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
Lowest Observed Effect Concentration (LOEC) based on biomass integral:0.125 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

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

Nominal Concentration (mg/l)		Cell Densities*(cells per ml)		Inhibition Values (%)
Nominal Concentration (mg/l)		0 Hours	72 Hours	Growth Rate	Yield/Biomass Integral
Control	R₁	4.98E+03	2.14E+05
	R₂	5.06E+03	1.92E+05	-	-
	Mean	5.02E+03	2.03E+05
Solvent Control	R₁	5.63E+03	3.30E+05
	R₂	4.35E+03	2.12E+05	-	-
	Mean	4.99E+03	2.71E+05
0.0027	R₁	4.18E+03	4.59E+05
	R₂	4.91E+03	4.16E+05	[15]	[63]
	Mean	4.54E+03	4.37E+05
0.027	R₁	5.45E+03	4.00E+05
	R₂	4.79E+03	2.47E+05	[5]	[20]
	Mean	5.12E+03	3.24E+05

[ ]

Table 2 Cell Densities and pH Values in the DefinitiveTest

0-Hour Mean Measured Test Concentration (mg/l)		pH	Cell Densities*(cells per ml)				pH
0-Hour Mean Measured Test Concentration (mg/l)		0 h	0 h	24 h	48 h	72 h	72 h
Control	R₁	7.4	3.54E+03	2.84E+04	1.23E+05	5.13E+05	8.0
	R₂	7.4	3.26E+03	2.67E+04	1.24E+05	3.42E+05	8.1
	R₃	7.4	4.91E+03	2.59E+04	7.46E+04	2.90E+05	8.1
	R₄	7.4	4.34E+03	2.94E+04	7.25E+04	3.01E+05	8.2
	R₅	7.3	3.52E+03	2.71E+04	8.42E+04	2.53E+05	8.2
	R₆	7.3	3.22E+03	3.26E+04	8.96E+04	2.52E+05	8.1
	Mean		3.80E+03	2.83E+04	9.46E+04	3.25E+05
Solvent Control	R₁	7.3	3.80E+03	3.21E+04	1.04E+05	2.94E+05	8.0
	R₂	7.3	3.42E+03	2.85E+04	1.03E+05	3.00E+05	8.0
	R₃	7.3	3.54E+03	3.09E+04	8.42E+04	2.87E+05	7.9
	R₄	7.3	3.38E+03	2.78E+04	9.20E+04	3.02E+05	7.9
	R₅	7.3	3.69E+03	2.39E+04	1.09E+05	3.19E+05	7.8
	R₆	7.3	3.86E+03	3.08E+04	9.52E+04	3.09E+05	7.9
	Mean		3.61E+03	2.90E+04	9.79E+04	3.02E+05
0.42	R₁	7.2	3.82E+03	2.28E+04	9.57E+04	3.31E+05	7.9
	R₂	7.2	3.99E+03	2.49E+04	9.37E+04	3.28E+05	7.8
	R₃	7.2	4.65E+03	2.10E+04	1.10E+05	3.20E+05	7.9
	R₄	7.2	4.10E+03	2.36E+04	9.41E+04	3.01E+05	7.8
	R₅	7.2	4.18E+03	2.47E+04	9.26E+04	2.87E+05	7.8
	R₆	7.2	4.68E+03	3.29E+04	8.75E+04	2.98E+05	7.8
	Mean		4.24E+03	2.50E+04	9.55E+04	3.11E+05

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		0.082		0.061		0.060
	R₂		0.079		0.064		0.042
	R₃		0.078		0.044		0.057
	R₄		0.083		0.038		0.059
	R₅		0.080		0.047		0.046
	R₆		0.087		0.042		0.043
	Mean		0.082		0.049		0.051
Solvent Control	R₁		0.087		0.049		0.043
	R₂		0.082		0.053		0.045
	R₃		0.085		0.042		0.051
	R₄		0.081		0.050		0.050
	R₅		0.075		0.063		0.045
	R₆		0.085		0.047		0.049
	Mean		0.083		0.051		0.047

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

0-Hour Mean Measured Test Concentration (mg/l)		Growth Rate (cells/ml/hour)		Yield (cells/ml)		Biomass Integral (cells/ml/hour)
0-Hour Mean Measured Test Concentration (mg/l)		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*	0 – 72 h	% Inhibition
Control	R₁	0.067		5.09E+05		9.54E+06
	R₂	0.062		3.38E+05		7.47E+06
	R₃	0.059		2.85E+05		5.65E+06
	R₄	0.060	-	2.97E+05	-	5.82E+06	-
	R₅	0.058		2.50E+05		5.47E+06
	R₆	0.058		2.48E+05		5.71E+06
	Mean	0.061		3.21E+05		6.61E+06
	SD	0.003		9.80E+04		1.61E+06
Solvent Control	R₁	0.060		2.90E+05		6.56E+06
	R₂	0.060		2.96E+05		6.50E+06
	R₃	0.059		2.84E+05		5.97E+06
	R₄	0.060	-	2.99E+05	-	6.26E+06	-
	R₅	0.061		3.16E+05		6.78E+06
	R₆	0.060		3.05E+05		6.49E+06
	Mean	0.060		2.98E+05		6.43E+06
	SD	0.001		1.13E+04		2.78E+05
0.42	R₁	0.061	[2]	3.27E+05		6.58E+06	[2]
	R₂	0.061	[2]	3.24E+05		6.55E+06	[2]
	R₃	0.061	[2]	3.16E+05		6.74E+06	[5]
	R₄	0.060	0	2.97E+05		6.20E+06	4
	R₅	0.059	2	2.83E+05		6.02E+06	6
	R₆	0.060	0	2.93E+05		6.23E+06	3
	Mean	0.060	[1]	3.07E+05	[3]	6.39E+06	1
	SD	0.001		1.82E+04		2.75E+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

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 based on the 0-Hour mean measured test concentration gave EC50 values of greater than 0.42 mg/l. Correspondingly the No Observed Effect Concentration was 0.42 mg/l.
Based on the geometric mean measured test concentrations the EC50 values were estimated to be greater than 0.32 mg/l. Correspondingly the No Observed Effect Concentration was 0.32 mg/l.
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:

A previous study conducted on this test material showed the substance was slightly soluble in water (38.1 mg/L). However the maximum dissolved test material concentration that could be obtained (by visual inspection) was 2.0 mg/L using a preliminary solution in methanol.

Based on this a pre-study media preparation trial was conducted which indicated that a dissolved test material concentration of approximately 0.027 mg/l was obtained from a solvent spike method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to an aqueous solution of the test material at a 0-Hour mean measured concentration of 0.42 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Based on the 0-Hour measured concentration obtained further inspection of the pre-study media preparation trial results would suggest that an error occurred in the preparation of the solvent spike sample which in turn resulted in the comparably low value obtained from the centrifuged test sample.

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 0.42 mg/l and correspondingly the No Observed Effect Concentration was 0.42 mg/l.

The test concentration of 0.42 mg/l was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent, and having due regard to the amount of auxiliary solvent permitted in the test under the OECD Guidelines.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.41 to 0.43 mg/l. A slight decline in measured test concentrations was observed at 72 hours in the range of 0.20 to 0.29 mg/l. Given that the preliminary stability analyses conducted indicated that the test material was stable over the test duration this decline was considered to be due to possible adsorption of the test material to the algal cells present. Whilst the preliminary recovery analyses conducted in the presence of algal cells indicated that no immediate adsorption occurred this does not preclude long-term adsorption over the test period. Adsorption was not a factor in the preliminary stability analyses conducted as no algal cells were present.

Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data.

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Chromium, 1-[2-[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]diazenyl]-2-naphthalenol 1-[2-[2-hydroxy-4(or 5)-nitrophenyl]diazenyl]-2-naphthalenol ammonium sodium complexes

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Chromium, 1-[2-[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]diazenyl]-2-naphthalenol 1-[2-[2-hydroxy-4(or 5)-nitrophenyl]diazenyl]-2-naphthalenol ammonium sodium complexes

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