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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 March 2012 to 09-August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Qualifier:
according to
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
During the final test singular samples for possible analysis were taken from all test concentrations and the control according to the following schedule:
- Frequency: at t=0 h, t=24 h and t=48 h
- Volume: 3 mL
- Storage: Samples were stored in a freezer until analysis.
- Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Additionally, singular reserve samples of 3 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Details on test solutions:
Preparation of test solutions:
The standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that would disturb the test system was prevented as much as possible (e.g. film of the test substance on the water surface). The batch of CP Formate tested was a clear colourless liquid with a purity of 94.1% and the substance was not completely soluble in test medium at the higher loading rates prepared. Preparation and weighing were as much as possible performed under dimmed light conditions. Test solutions at loading rates of 1 mg/L and higher were all individually prepared. Preparation included one day of magnetic stirring followed by a settlement period of 1 hour. This generally resulted in a clear and colourless solution with some floating material at loading rates of 10 mg/L and higher. The Water Accommodated Fraction (WAF) was then collected from the middle of the solutions thus removing the floating material from the end test solutions. The solutions without a floating layer were used as such. All final test solutions were clear and colourless. Not e that in addition a ten-fold dilution was prepared from the 1 mg/L solution for testing in the combined limit/range-finding test. All was performed in closed units as to minimise any possible loss due to the expected volatile character of the test substance. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 1E+04 cells/mL.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM:
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Reason for selection:This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.

FRESH WATER ALGAE CULTURE:
-Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
- Light intensity: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
-Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford,Mass., USA).
- Acclimation period: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Culturing media and conditions: M2; according to the OECD 201 Guideline, formulated using Milli-Q water (tap water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges: Milli-Q water; Millipore Corp.,Bedford, Mass., USA) preventing precipitation.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
based on the expected volatile character of the test substance the test period was shorter than the standard of 72 hours
Hardness:
Hardness (Ca + Mg): 0.24 mmol/l (24 mgCaCO3/l)
Test temperature:
21.9-23.3 °C
pH:
t=0h: 7.4-7.5
t=48h: 8.3-9.9
Dissolved oxygen:
No details in study report
Salinity:
No details in study report
Nominal and measured concentrations:
- Nominal concentrations: 1.0 and 3.2 mg/L and WAFs prepared at loading rates of 10, 32 and 100 mg/L.
- Analytical concentrations (time weghted average t=0, 24, 48h): 0.19, 0.49, 1.4, 4.5 and 7.1 mg/L
Details on test conditions:
- Test duration: 48 hours (based on the expected volatile character of the test substance the test period was shorter than the standard of 72 hours, which is acceptable as long as unlimited growth and a multiplication factor of at least 16 are met).
- Test vessels: 100 mL, all-glass, airtight closed using an aluminium screwcap with septum and containing 75 mL of test solution.
- Medium: M2
- Cell density: An initial cell density of 1E+04 cells/mL.
- Illumination: Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 93 to 100 µE.m-2.s-1.
- Incubation: Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
- Replicates: 3 replicates of each test concentration, 6 replicates of the control, 1 or 2 replicates of each concentration without algae for sampling after 24 hours.

Appearance of the cells:
At the end of the final test microscopic observations were performed on the two highest test groups to observe for any abnormal appearance of the algae.

Recording of cell densities:
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 7.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
1.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Reduction of growth rate gradually increased with increasing TWA concentration from 1.4% at the lowest to 43% at the highest concentration. Statistically significant reduction of growth rate was found at test concentrations of 0.49 mg/L and higher (Bonferroni t test, α = 0.05).
Inhibition of yield increased with increasing TWA concentration of CP Formate from 0.19 mg/L upwards resulting in 83% inhibition at a TWA concentration of 7.1 mg/L. Statistically significant inhibition of yield was found at TWA concentrations of 0.49 mg/L and higher (Bonferroni t test, α = 0.05).
Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
Results with reference substance (positive control):
The EC50 for growth rate reduction (ERC50: 0-72h) was 0.98 mg/L with a 95% confidence interval ranging from 0.74 to 1.3 mg/L. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/L. Hence, the ERC50: 0-72h for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (EYC50: 0-72h) was 0.44 mg/L with a 95% confidence interval ranging from 0.32 to 0.61 mg/L. The historical ranges of the 72h EC50 for yield inhibition lie between 0.43 and 1.1 mg/L. Hence, the EYC50: 0-72h for the algal culture tested corresponds with this range.
Reported statistics and error estimates:
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant reduction of growth rate or inhibition of yield (ANOVA, Bonferroni t-test, TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman). Additionally, the EC10 was determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993. Calculation of the EC50 and EC10 values was based on log-linear regression analysis of the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test substance.
Validity criteria fulfilled:
yes
Remarks:
See 'Any other information on materials and methods incl. tables'
Conclusions:
The 48-h ErC50 and 48-h ErC10 are 7.1 mg/L and 1.5 mg/L, respectively in green algae.
Executive summary:

A GLP-compliant OECD 201 guideline study with freshwater algae (Desmodesmus subspicatus) is available for CP Formate. Algae were exposed to nominal concentrations of 1.0 and 3.2 mg/L and WAFs prepared at loading rates of 10, 32 and 100 mg/L under static conditions for 72 hours. Because the test material is volatile, testing was conducted in airtight closed test vessels in order to minimise possible losses due to volatilisation. At the start of the test, the actual test concentrations were 0.81, 2.3, 7.7, 23 and 35 mg/L for the concentrations prepared at 1.0, 3.2, 10, 32 and 100 mg/L, respectively. The measured concentrations decreased to 10-17% of initial after 24 hours and further to 1.7-5.5% of initial after 48 hours of exposure. Based on these results, the Time Weighted Average (TWA) exposure concentrations were calculated. The TWA concentrations corresponded with 0.19, 0.49, 1.4, 4.5 and 7.1 mg/L.

Reduction of growth rate gradually increased with increasing TWA concentration from 1.4% at the lowest to 43% at the highest concentration. Statistically significant reduction of growth rate was found at test concentrations of 0.49 mg/L and higher. Inhibition of yield increased with increasing TWA concentration of CP Formate from 0.19 mg/L upwards resulting in 83% inhibition at a TWA concentration of 7.1 mg/L. Statistically significant inhibition of yield was found at TWA concentrations of 0.49 mg/L and higher. Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control. Based on these data, the 48 -h EC50 values for growth and yield were determined to be >7.1 mg/L and 2.0 (95% C.L: 0.86 -4.8 mg/L), respectively. The 48 -h ErC10 and EyC10 values were determined at 1.5 mg/L and 0.27 mg/L, respectively. The 48 -h NOEC finally, is 0.19 mg/L for both endpoints. The validity criteria were not met in this study as the pH in various solutions during both the combined limit/range-finding and the final test increased with more than 1.5 units and above 9.0 at the end of the test. The increase was due to the volatility of the test substance and the consequence of testing in a closed system. Despite the addition of extra NaHCO3 and the adjustment of the pH of the test medium before the study it proved not possible to maintain the pH. However, algal growth met the criterion of at least a 16-fold increase. Altogether, the study integrity is considered to be not adversely affected by the deviation.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See 'Attached justification'
Reason / purpose:
read-across source
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
7.7 mg/L
Remarks on result:
other: as determined from read-across to CP Formate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.6 mg/L
Remarks on result:
other: as determined from read-across to CP Formate

Description of key information

The toxicity to aquatic algae is assessed based on read-across from the close structural analogue CP Formate (CAS# 25225 -08 -5) and the ErC50 value and ErC10 values determined at 7.7 mg/L and 1.6 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
7.7 mg/L
EC10 or NOEC for freshwater algae:
1.6 mg/L

Additional information

A GLP-compliant OECD 201 guideline studywith freshwater algae (Desmodesmus subspicatus)is available for CP Formate. Algae were exposed to nominal concentrations of 1.0 and 3.2 mg/L and WAFs prepared at loading rates of 10, 32 and 100 mg/L under static conditions for 72 hours. Because the test material is volatile, testing was conducted in airtight closed test vessels in order to minimise possible losses due to volatilisation. At the start of the test, the actual test concentrations were 0.81, 2.3, 7.7, 23 and 35 mg/L for the concentrations prepared at 1.0, 3.2, 10, 32 and 100 mg/L, respectively. The measured concentrations decreased to 10-17% of initial after 24 hours and further to 1.7-5.5% of initial after 48 hours of exposure. Based on these results, the Time Weighted Average (TWA) exposure concentrations were calculated. The TWA concentrations corresponded with 0.19, 0.49, 1.4, 4.5 and 7.1 mg/L.

Reduction of growth rate gradually increased with increasing TWA concentration from 1.4% at the lowest to 43% at the highest concentration. Statistically significant reduction of growth rate was found at test concentrations of 0.49 mg/L and higher. Inhibition of yield increased with increasing TWA concentration of CP Formate from 0.19 mg/L upwards resulting in 83% inhibition at a TWA concentration of 7.1 mg/L. Statistically significant inhibition of yield was found at TWA concentrations of 0.49 mg/L and higher. Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control. Based on these data, the 48 -h EC50 values for growth and yield were determined to be >7.1 mg/L and 2.0 (95% C.L: 0.86 -4.8 mg/L), respectively. The 48 -h ErC10 and EyC10 values were determined at 1.5 mg/L and 0.27 mg/L, respectively. The 48 -h NOEC finally, is 0.19 mg/L for both endpoints. The validity criteria were not met in this study as the pH in various solutions during both the combined limit/range-finding and the final test increased with more than 1.5 units and above 9.0 at the end of the test. The increase was due to the volatility of the test substance and the consequence of testing in a closed system. Despite the addition of extra NaHCO3 and the adjustment of the pH of the test medium before the study it proved not possible to maintain the pH. However, algal growth met the criterion of at least a 16-fold increase. Altogether, the study integrity is considered to be not adversely affected by the deviation.