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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to OSPAR draft protocol for HOCNF, in compliance with GLP requirements
Qualifier:
equivalent or similar to
Guideline:
ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)
Version / remarks:
draft protocol at the time of testing
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Duplicate aliquots (4 ml) of solution or algal cell suspension from the range finding and definitive tests were mixed with Quickszint 1 scintillation fluid (Zinsser Analytic) prior to determination of radioactive content by liquid scintillation counting as a gel. Duplicate aliquots (up to 1 ml) of other liquid samples were mixed with Quickszint 1 scintillation fluid prior to determination of radioactive content by liquid scintillation counting as a liquid.
Vehicle:
no
Details on test solutions:
Appropriate amounts of radiolabelled and non-radiolabelled DBT were combined in methanol. The solutions were transferred into 1 litre capacity glass flasks and methanol removed under a stream of gaseous nitrogen. Each flask was amended with 1 litre algal medium and subjected to ultrasonication (20 min) followed by continuous stirring (18-24 h). For the range finding and limit tests, stock suspensions were prepared by adding 1 litre of algal medium to 45.4 µg [14C]-DBT and subjecting the mixture to ultrasonication (20 min) followed by continuous stirring (18-24 h). The suspension was centrifuged (1000 r.p.m., 30 min) using a Jouan Model C412 bench centrifuge and total radioactivity measured in the supernatant fraction. In the range finding test, the supernatant fraction was diluted as required with algal growth medium to produce the lower test concentrations.
Test organisms (species):
Skeletonema costatum
Details on test organisms:
The marine diatom Skeletonema costatum (Chrysophyta) was used for these studies. Starter cultures (Strain 1077/3) were obtained from the Culture Collection of Algae and Protozoa (CCAP), Oban, Scotland.

Transfers of the algae were made regularly into fresh algal growth medium to provide suitable axenic subcultures which were in the exponential phase of growth, for test inoculations.
Test type:
static
Water media type:
saltwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
no
Hardness:
not relevant (marine water)
Test temperature:
21.2 ± 1°C throughout the test period
pH:
7.97-8.45 during the test
Dissolved oxygen:
no data
Salinity:
Algal growth medium (Guillard's Medium - Appendix 2) was prepared by adding specific weights/volumes of nutrient enrichments to 0.2 µm filtered natural seawater. The seawater was collected from a site on the east coast of Scotland and was free from known sources of pollution. The seawater typically has a salinity of 32 ± 2% and a pH of 8.0 ± 0.3.
Nominal and measured concentrations:
For limit test initial measured concentration was 16 µg/L. After 72h exposure, concentrations measured in the 6 test flasks ranged from 4.2 to 6.0 µg/L with an average of 5.3 µg/L
Details on test conditions:
For the range finding test, test concentrations were prepared by dilution of the supernatant fraction with algal medium. The test was conducted, over a 72 h period, using the following series of measured concentrations of test material in algal growth medium, inoculated with 10E3-10E4 cells/ml Skeletonema costatum:
17.2, 3.5, 0.9 and 0.2 µg equiv/L
A control was also set up using algal growth medium alone.
A further control was set up using algal growth medium alone using flasks which had not been treated with Sigmacote®.
Two flasks were prepared at each concentration.
A further single flask was prepared at each concentration which was not inoculated with algae, to provide information of the bio-availability of the test material.
Each flask contained 50 ml of test solution.

Based on the results from the range finding test, a limit test was conducted at the maximum solubility [14C]-DBT.
For the limit test, 10 flasks containing test solution at maximum solubility prepared as described above and 6 flasks containing algal growth medium alone were tested. The concentration of radioactivity in the supernatant fraction was measured as 16.0 µg equiv/L. An additional 3 control flasks which had not been treated with Sigmacote® were set up using algal growth medium alone.
Each flask contained 50 ml of test solution.

The inoculum volume was calculated to yield 10E3-10E4 cells/mL Skeletonema costatum.
Two further single flasks containing test solution or algal growth medium alone were set-up and not inoculated with algae to provide information on the bioavailability of the test material.

Algal cell concentrations were measured in the inoculum and in each test flask at 72 h after the commencement of the range finding test. Algal cell concentrations were measured in the inoculum and in each algal flask at 24, 48 and 72 h after the commencement of the limit test. Aliquots were removed by pipette and cell concentrations determined using a Compound Light Microscope and Improved Neubauer Counting Chambers. Between 4 and 6 cell counts were made from each flask at each timepoint.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 16 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 16 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The deviation between µ(ave) gave results over 0-72 h for the cultures incubated with [14C]-DBT at its maximum solubility and the corresponding controls was very small (<2%). This achieved statistical significance, a result which is judged to reflect low variability between replicates within these treatment groups. No other statistically significant differences were found and it was concluded that the No Observed Effect Concentration (NOEC) and the EC50 value exceed the measured values found in the limit test.
Results with reference substance (positive control):
In accordance with UK MAFF Guidelines (1993), a reference toxicant (3,5-dichlorophenol at 1.5 µg equiv/L) was included as part of the study. Three flasks containing reference toxicant, inoculated with the same concentration of Skeletonema costatum and maintained under the same conditions as the test flasks, showed a 62% inhibition of growth over the 72 h test period. As this fell within the acceptance criteria of 20-80% inhibition as specified by UK MAFF, the test is considered to have been valid.
Validity criteria fulfilled:
yes
Remarks:
reference substance
Conclusions:
No EC50 or NOEC could be determined as no inhibition of growth was observed at highest soluble concentration (16 µg/L).
Executive summary:

Study of inhibition of growth of the marine algae Skeletonema costatum was carried out according to the protocol described in OSPAR document (now ISO 10253 standard). No significant inhibition was observed at the highest soluble (measured) concentration, which was 16.0 µg/L

Description of key information

No growth inhibition of algae is observed at the highest soluble concentration.

Key value for chemical safety assessment

Additional information

Several studies are available but for 4 out of 5 there is uncertainty on exposure concentration and/or use of vehicle in conditions where interference cannot be excluded. Only one study fulfills confidence criteria. It makes use of marine unicellular algae, but their conclusions will also be applied to freshwater, as indicated in the Guidance. This study (Inveresk, 1997) has been carried out on Skeletonema costatum, use was made of a 14C labelled test substance sample that allowed to have precise measurement of exposure concentrations. At the highest soluble concentration of 16 µg/L there was no difference of growth with the controls. This study is considered to be reliable and the key study.