Hatcol 1760

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 13 March 2008 and 28 March 2008.

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 relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). Date of inspection: 21/08/2007. Date of signature: 15/10/2007.

Test material

Test material information

Constituent 1

Details on test material:

Sponsor's identification :Hatcol 1760
Description : A clear colourless slightly viscous liquid
Batch number : A25983
Date received : 4 February 2008
Storage conditions :Room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Sample Preparation
Following the addition of sodium chloride (90 g) to the test sample (300 ml), it was extracted with dichloromethane (3 x 50 ml). The extracts were filtered through anhydrous sodium sulphate. The combined extracts were evaporated to dryness and the residue re-dissolved in acetonitrile (10 ml).

- Concentrations: 100 mg/l

- Sampling method: Samples were taken from the control (replicates R1 - R6 pooled) and the 100 mg/l loading rate WAF test group (replicates R1 - R3 and R4 - R6 pooled) at 0 and 72 hours for quantitative analysis. The test material concentration in the test samples was determined by high performance liquid chromatography (HPLC) using an external standard. The test material gave a chromatographic profile consisting of 10 peaks. The results have been calculated using the total peak area associated with the test material.



- Sample storage conditions before 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.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Validation of mixing period
Pre-study work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF. A WAF with a nominal loading rate of 100 mg/l was prepared, in duplicate, in culture medium. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the aqueous phases were then removed by siphon and the concentration of the test material in the 100 mg/l loading rate WAFs was verified by chemical analysis. Observations made on the WAF which had stirred for a period of 95 hours indicated that gross mixing of the test material had occurred. It was therefore considered appropriate to filter the aqueous phase through a glass wool plug prior to sampling for chemical analysis.

Due to the low aqueous solubility and complex nature of the test material for the purposes of the definitive test the test material was prepared as a Water Accommodated Fraction (WAF).
An amount of test material (250 mg) was dispensed onto the surface of 2.5 litres of culture medium via a plastic disposable syringe 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. This was stirred for 23 hours. 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 100 mg/l loading rate WAF was inoculated with algal suspension (42.5 ml) to give the required test concentration of 100 mg/l
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 72 hours (see Appendix 2).


- Eluate: same as culture media

- Differential loading: No

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

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable

- Evidence of undissolved material (e.g. precipitate, surface film, etc): 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 medium column with an oily layer/film of test material floating at the media surface. Microscopic examination of the WAF showed there to be no globules or micro-dispersions of test material to be present.

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 (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): not stated in report
- Method of cultivation: Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 104 - 105 cells/ml.


ACCLIMATION
- Acclimation period: not stated in report
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

All test and control cultures were inspected microscolically at 72 hours.

Test conditions

Hardness:

Not stated in report

Test temperature:

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

pH:

7.4 -7.5 for test material exposure at 0 hours, 7.5 - 7.6 for control at 0 hours

7.7 for test material and 7.8 for control at 72 hours

Dissolved oxygen:

Not stated in report

Salinity:

Not stated in report

Nominal and measured concentrations:

Range-finding test: 10 and 100 mg/l loading rate WAFs
Main test: 100 mg/l loading rate WAF

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass conical flasks
- Type: closed - plugged with polyurethane foam bungs to reduce evaporation
- Material, size, headspace, fill volume: 250 ml glass conical flasks each containing 100 ml of test preparation
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable, as static test conditions
- Renewal rate of test solution (frequency/flow rate): not applicable, as static test conditions
- Initial cells density: initial cell density of approximately 10^3 cells/ml
- Control end cells density: 1.2 x 10^5 cells/ml
- No. of organisms per vessel: not applicable
- No. of vessels per concentration (replicates): Six flasks each containing 100 ml of test preparation were used for the 100 mg/l loading rate WAF treatment group..
- No. of vessels per control (replicates): . Six flasks each containing 100 ml of test preparation were used for the control
- No. of vessels per vehicle control (replicates): not applicable


GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: not applicable


TEST MEDIUM / WATER PARAMETERS
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.


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: not required
- Photoperiod: constant illumination
- Light intensity and quality: not stated in report
- Salinity (for marine algae): not applicable


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- 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: No



TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable, as limit test
- Justification for using less concentrations than requested by guideline: no effects observed in range-finding study
- Range finding study
- Test concentrations: 10 and 100 mg/l
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at the 10 and 100 mg/l loading rate WAFs.
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.

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:

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

- Observation of abnormalities (for algal test):
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

- Any stimulation of growth found in any treatment: No

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Microscopic examination of the WAF showed there to be no globules or micro-dispersions of test material to be present.

- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes

- EC50: Accordingly the following results were determined from the data:
ErC50 (0 - 72 h) : 0.64 mg/l
EyC50 (0 - 72 h) : 0.36 mg/l; 95% confidence limits 0.30 - 0.43 mg/l
EbC50 (0 - 72 h) : 0.32 mg/l; 95% confidence limits 0.29 - 0.35 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: 0.125 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
Lowest Observed Effect Concentration (LOEC) based on biomass integral : 0.25 mg/l

The results from the positive control with potassium dichromate were within the normal range 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

Pre-study work indicated that there was a significant increase in the measured test concentrations by extending the preparation period from 24 to 96 hours. However, observations made on the WAF stirred for a period of 95 hours indicated that gross mixing of the test material had occurred. Whilst the aqueous phase was removed and filtered through a glass wool plug it was considered that some undissolved test material may have remained which may have resulted in excessively high measured test concentrations. As such it was considered that a preparation period of 24 hours was sufficient to ensure equilibration between the test material and water phase.

 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 (please see attached).

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

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.

DefinitiveTest

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 (please see attached). 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 (please see attached).

 Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 70 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                            :   3.87 x 10³cells per ml
Mean cell density of control at 72 hours                          :   2.71 x 10⁵cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 23% 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

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

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

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

where E_rL*_xis 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) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences (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.

Inhibition of yield

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

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

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

where E_yL*_xis the loading rate that reduced yield by x%.

There were no statistically significant differences (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 yield was 100 mg/l loading rate WAF.

Inhibition of biomass integral

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

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

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

where E_bL*_xis the loading rate that reduced biomass integral by x%.

There were no statistically significant differences (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 biomass integral was 100 mg/l loading rate WAF.

*EL = Effective Loading rate

*EL = Effective Loading rate

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Desmodesmus subspicatus to the test material gave EL*50 values of greater than 100 mg/l loading rate WAF and 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 Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

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.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Desmodesmus subspicatus was exposed to an aqueous solution of the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

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

Results. Exposure of Desmodesmus subspicatus to the test material gave 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 and 72 hours showed measured test concentrations to be less than the limit of quantitation (LOQ) of the analytical method employed which was assessed down to 0.69 mg/l.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole, and the dissolved test material was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

Exposure of Desmodesmus subspicatus to the reference material, potassium dichromate, gave an E_rC₅₀ (0 - 72 h) of 0.64 mg/l*, an E_yC₅₀ (0 - 72 h) of 0.36 mg/l; 95% confidence limits 0.30 ‑ 0.43 mg/l, and an E_bC₅₀ (0 - 72 h) of 0.32 mg/l; 95% confidence limits 0.29 - 0.35 mg/l. The Lowest Observed Effect Concentration based on inhibition of growth rate, yield and biomass integral was 0.25 mg/l and the No Observed Effect Concentration was 0.125 mg/l. 

*EL = Effective Loading Rate

*It was not possible to calculate 95% confidence limits for the E_rC₅₀ value as the data generated did not fit the models available for the calculation of confidence limits.