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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:
2-5 July 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline & GLP study. Analytical method validation is lacking, the composition given in appendix but batch no not identical to supplier's batch no., no phys-chem data, however the final conclusion is not influenced and therefore is valid. Due to the use of a vehicle, the results are questionable wrt actual situation when acetone is not involved. pH increase > 1.5 units, no reference substance, no range finder, no analysis of variation of the controls. NOEC value should be >= value, report mentions <=.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
no reference substance, no range finder, no analysis of variation of the controls.
Principles of method if other than guideline:
Guideline version 1984 was used.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The concentrations of Aquapel 364 in the test solutions were measured at 0 and 72 hours using a high performance liquid chromatography method. On each day of analysis, the sample preparation procedure was validated by fortifying triplicate 20 ml aliquots of culture medium with 50 ul of a 400 mg L-1 stock of Aquapel 364, prepared in acetone. These 1.0 mg L-1 fortification samples were taken through the analytical procedure
Vehicle:
yes
Details on test solutions:
This study was run with a culture medium and a solvent control together with a nominal Aquapel 364 concentration of 1.0 mg I-1.
The test substance was of low solubility, therefore, stock solutions in a solvent were prepared. A stock concentrate of Aquapel 364 was prepared by dispersing 0.1 g of Aquapel 364 in 10 ml of acetone. The test solution was prepared from this stock solution by addition of appropriate volumes to 1 L of culture medium and stirring thoroughly. All additions were made sub-surface, gradually, by microlitre syringe, into solutions vigorously stirred by magnetic follower. The resultant solution after 5 minutes ultrasonication, was clear and colourless with afew very fine white particulates oin a homogenous suspension.
A solvent control was similarly prepared by the addition of 0.1 ml of acetone to 1 litre of culture medium, to give a solvent concentration of 100 ul/L. The control consisted of culture medium only.

Using aseptic techniques, 100 ml volumes of test solution were dispensed to each test vessel and 150 ml to each blank vessel, with the remainder of the test solutions being used for physical and chemical analyses on day 0.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test species was the unicellular green alga Selenastrum capricornutum ATCC 22662 from laboratory cultures maintained under axenic conditions. A 3 day old culture of the alga in the exponential growth phase was used as inoculum for the test. The culture was grown in the medium, and under the environmental conditions, described for the test.
A full description of the algal medium is attached to the report as appendix, but is in line with the US EPA medium constituents.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
See standard culture medium
Test temperature:
24.0 - 24.1 C
pH:
7.4-7.5 at the start of the test
9.1-9.7 at the end of the test
Dissolved oxygen:
No data
Nominal and measured concentrations:
Nominally: control-solvent control-1.0 mg/L
Actual: control and solvent control: below limit of detection
1.0 mg/L: t= 0 : 0.41 mg/L, t =72h : below limit of detection
Details on test conditions:
The test vessels were borosilicate glass conical flasks of 250 ml nominal capacity closed with polyurethane foam bungs. Each flask contained 100 ml of test solution. The cultures were incubated at 24 +/- 1°C (the nominal test temperature), under continuous "cool-white" illumination, with orbital shaking at 160 rpm, in a Gallenkamp type INR-401 orbital incubator.
Six replicate cultures of the culture medium control, solvent control and each concentration of Aquapel 364 were employed. The positions of the test vessels in the incubator were randomised by rows, and re-randomised daily. One blank vessel (without algal inoculum) for each control and test concentration was incubated concurrently.
The algal cell densities of the inoculum and test cultures were determined by electronic particle counting, using a Coulter counter model ZM, counting at a lower threshold equivalent spherical diameter of approximately 2.3 um. Each replicate test vessel was inoculated with 0.600 ml of the inoculum culture to give a nominal cell density of 1.00 x 10^4 cells ml-1. Three 100 ml volumes of Coulter electrolyte, inoculated in the same manner, had a mean measured cell density of 1.01 x 10^4 cells ml-1. The latter value was used for growth calculations.
After 24, 48 and 72 hours, samples were removed from each test and blank vessel. The appropriate blank particle count was subtracted from that of the test culture to obtain the cell density.

The pH of each test solution was measured at the start of the test, with a calibrated pH meter, using the excess remaining after filling the test vessels. At the end of the test the pH of two of the replicate test solutions (containing algae) from the culture medium and solvent controls and single test concentration was determined. The temperature of the incubator was measured daily by a thermometer calibrated to 0.1°C, and was continuously monitored, with hourly recording of values, using an electronic recording system. The light intensity was measured once during the study, using Skye Instruments photometers reading in lux (7830 lux) and quantum units (95.0 uEinstein m-2 s-1).
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EL50
Remarks:
EbC50
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EL50
Remarks:
ErC50
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Details on results:
No significant effects on growth rate or growth were observed.
An increase factor in algal cell density of 344 was observed.
Reported statistics and error estimates:
The areas under the growth curve were examined by one-way analysis of variance, and Dunnett's procedure was used to identify significant differences (P=0.05) from the solvent control.
The percentages of the solvent control were transformed to probability scale and analysed by linear regression against log concentration, to estimate the median effective concentration (based on biomass curve area, EbC50) and its 95% confidence limits.

 

Nominal concentration of Aquapel 364 (mg/L)

Mean area under growth curve (0-72 h)

Percentage of solvent control

Culture medium control

232.6

99

Solvent control

235.1

-

1.0

234.2

100

 

Nominal concentration of Aquapel 364 (mg/L)

Mean growth rate (0-72 h)

Percentage of solvent control

Culture medium control

1.946

100

Solvent control

1.949

-

1.0

1.942

100

 

Validity criteria fulfilled:
no
Remarks:
according to guideline 2006, coefficients of variation can not be calculated
Conclusions:
Guideline study which can be repeated, however analysis do not contribute to the understanding of concentration applied. Some phys-chem data are missing (solubility, vapour pressure), batch no. of supplier and composition appendix do not match. pH increase > 1.5 units, no reference substance, no range finder, no analysis of variation of the controls. NOECs not <= but >= values. Conclusion should be based on nominal conc. Use of acetone as vehicle makes extrapolation to actual situation (without solvent) questionable. Reliability 2, Relevance and adequacy doubtful.
Executive summary:

In this study, the freshwater alga Selenastrum capricornutum was exposed to Aquapel 364 for 72 h according to OECD guideline 201. Control, solvent control and 1.0 mg/L dose groups were tested in six replicates in a static test design. Aquapel 364 was added to the test medium after being dissolved in acetone. Algal cell densities were determined every 24 hours. Growth and growth rate were calculated, but no significant differences could be established. Based on nominal concentrations, the EC50 for both growth (EbC50) and growth rate (ErC50) were > 1.0 mg/L. The NOEC was determined at >= 1.0 mg/L. Analysis of the actual concentrations showed a mean measured concentration of Aquapel 364 of < 7% to 41% of the nominal concentration. Only at t=0 h Aquapel 364 could be determined by analysis.

Description of key information

Key study is Brixham Environmental laboratories (2002). OECD201, Selenastrum capricornutum, static 72 h. Use of vehicle (acetone). Study considered reliable with restrictions. No statistically significant growth effects were observed. End-point concluded on measured concentrations (with the aid of acetone) will not give reliable estimate for derivation of PNEC, since acetone will not be used under normal conditions. Based on mean measured concentrations the ErC50 will be > 0.17 mg/L and NOErC >= 0.17 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.17 mg/L
EC10 or NOEC for freshwater algae:
0.17 mg/L

Additional information

Key study is Brixham Environmental Laboratory (2001). No statistically significant growth effects were observed. End-point concluded on measured concentrations (with the aid of acetone) will not give reliable estimate for derivation of PNEC, since acetone will not be used under normal conditions. Based on mean measured concentrations the ErC50 will be > 0.17 mg/L and NOErC >= 0.17 mg/L.


 


Composition given in appendix but batch no not identical to supplier's batch no. Phys-chem data and analytical method validation is lacking, and due to the use of a vehicle, the results are questionable wrt actual situation when acetone is not involved. However, the study is performed according to Guide Lines and GLP and is considered reliable with restrictions.