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Ecotoxicological information

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:
From 30-06-2015 till 03-07-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP, All validity criteria fulfilled, idenfication of test substance complete, including chemical analyses
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
The NaHCO3 concentration of the test medium was increased to 150 mg/L to maintain a more constant pH. This small change will have no influence on the test result.
Principles of method if other than guideline:
Test medium
The test medium (OECD algae medium) was prepared according to the OECD and EC guideline (OECD, 2011 and Commission Regulation, 2008) The NaHCO3 concentration of the test medium was increased to 150 mg/L to maintain a more constant pH. This small change will have no influence on the test result.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Sampling
Samples were taken in the normal manner (water samples) at the start and end of the test from all concentrations and the control replicate. Samples were diluted 1:1 with leaching solution prior to analysis.
At least 10 mL was sampled in each case. Samples containing algae were filtered using a 0.45 μm filter to remove algae prior to analysis. Filters were primed with the relevant solution before use. Further dilution to within the range of the calibration curve was then carried out as required in leaching solution and samples were then added to HPLC vials. The samples were analyzed as soon as possible after sampling no storage took place.

Physico-chemical parameters and purity of algae
The pH of all samples and controls were measured at the beginning (t=0h) and at the end (t=72h) of the test. The temperature in the culturing apparatus was continuously measured and read out at the end of the test. The light intensity was measured at the beginning and at the end of the test. At the end of the test five random samples were microscopically checked for purity of the algal culture.

Chemical analyses
Chemical analyses were performed according to the analytical procedure described in Annex 7. The chemical analyses were performed on all samples taken. All components were measured. Data on the main component was not provided by the sponsor. An average recovery based on all three
measured components was therefore used for expression of the toxicological endpoints.
Vehicle:
no
Details on test solutions:
Test solutions
Preparation of the stock solutions
The test substance is sufficiently soluble to dilute the test concentrations from a stock solution. To prepare the stock solution 0.0019 g of test substance was weighed on an analytical balance 100 mL OECD medium was added and then ultrasonically treated for 1minute on ice. The stock appeared
homogeneous without visible particles. This stock was then discarded as it was intended to pre-treat glassware. It was then remade in the same manner with the same glassware by weighing in 0.0013g of test substance. The temperature indicated by the sonication apparatus was 35.1ºC. The stock was allowed to cool while stirring prior to use for preparing test solutions.
The pH of the stock solution was checked and found to be 7.9 and was therefore not adjusted.
Preparation of the test solutions
The test solutions were prepared in triplicate by addition of the adequate amounts of stock solution to the test vessels. Then test medium was added up to the required final volume. Six control vessels containing only test medium were included in the test.
An additional fourth sacrificial replicate was also made in the same manner for analytical purposes at the start and end of the test due to the sizes of the samples required and a further two vessels at 0.11 and 0.53 mg/L were made for a full extraction at the start of the test.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Source and maintenance of algae
The test was carried out with the freshwater unicellular algae Pseudokirchneriella subcapitata (CCAP 278/4) obtained from the Culture Collection of Algae and Protozoa SAMS Research Services Ltd.
Dunstaffnage Marine Laboratory, Dunbeg, Argyll, Scotland. After purchasing this strain was cultured and maintained. Cultures on sloped agar tubes were stored at 4°C in the dark until required.
Exponentially growing cultures are maintained at 23 ± 2°C in a temperature-controlled illuminated orbital incubator and are re-cultured under sterile conditions weekly to keep the algae in this phase.
The sensitivity of the algae was checked by performing a growth inhibition test with a reference compound (potassium dichromate) twice a year , and is not used unless found to be between the set EC50 values of 0.25 to 2.0 mg/L as indicated in the study guideline.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Standard OECD test medium, the NaHCO3 concentration of the test medium was increased to 150 mg/L
Test temperature:
The temperature varied from 21.6 to 23.3 °C during the test.
pH:
The pH measurements in Annex 3 show a maximum increase of 0.6 pH units.
Dissolved oxygen:
The test vessels were rotated continuously at a speed sufficient to prevent sedimentation of the algae.
Salinity:
Standard OECD test medium, the NaHCO3 concentration of the test medium was increased to 150 mg/L
Nominal and measured concentrations:
nominal test concentrations: 0.05, 0.11, 0.24, 0.53, and 1.17 mg/L.
Geometric Mean Concentrations: 0.008, 0.012 0.017, 0.025, 0.038 mg/L
Details on test conditions:
The test was performed in 100 mL Erlenmeyer flasks containing 40 mL of medium. The test flasks were closed with cotton-wool stoppers. Vessels to be extracted had cut glass necks to allow complete sealing of the vessel during extraction.

Culturing cabinet and test conditions
The test was carried out in a temperature-controlled illuminated orbital incubator, in which the temperature was maintained at 23 ± 2°C, and a continuous uniform illumination was provided in the spectral range of 400 to 700 nm by using fluorescent lamps at a distance of about 0.36 ± 0.02 m from
the algal cultures. The light intensity was in the range of 60 to 120 μE·m-2·s-1 (= μmol.m-2·s-1). The test vessels were rotated continuously at a speed sufficient to prevent sedimentation of the algae.

Test medium
The test medium (OECD algae medium) was prepared according to the OECD and EC guideline (OECD, 2011 and Commission Regulation, 2008) The NaHCO3 concentration of the test medium was increased to 150 mg/L to maintain a more constant pH. The test medium was prepared from concentrated solutions of the mineral salts prepared in deionized water and filter sterilized.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.05 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.23 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 0.11 - 0.527
Details on results:
Significant effect (growth rate) was observed on growth rate at 0.04 mg/L. The NOEC is therefore <0.04 mg/L and LOEC is 0.04 mg/L. The ErC10 is 0.050 mg/L and the ErC50 is 0.23 mg/L. The EbC50 was found to be 0.034 mg/L.

The following quality criteria have been met in the present study:
• The cell density of the controls increased at least a factor 16 within 72 hours.
• The mean coefficient of variation for section-by-section specific growth rates in the control cultures must not exceed 35%.
• The coefficient of variation of average specific growth rates during the whole test period in the replicate control cultures should not exceed 7%.
• Analytical quality criteria were met.
• Algae strain used was of acceptable sensitivity.


The concentrations measured during the test were not maintained. This is not uncommon for adsorbing substances due to their tendency to bind to organic matter. For this reason an extraction of the test vessels was also carried out to determine if the start and end of test recovery could be improved, explained or at least demonstrate likely binding to algae cells should the extraction method not be 100% efficient.

The total extractions that took place at the start of the test (T=0) gave almost identical recoveries to the normal sampling method and delivered no additional information or indication of why the recovery at the start of the test had fallen from 100% to approximately 79.9 - 83.6 %.

Preliminary investigations and information from the sponsor indicated that extraction was more efficient when using acidified leaching solution. For this reason acidified leaching solution was used at the end of the test by study plan amendment. Despite one anomalous result an acidified extraction method at the end of the test did appear to result in increased recovery of the test material. Supporting in part the belief that the test substance is still present but bound to the algae and largely non extractable with existing methods. Further investigation to more severe extraction methods could be advantageous here to demonstrate complete exposure to the test substance. Although data for the extractions is limited in this study, they have however demonstrated that the ½ LOD or LOQ approach sometimes used if the end concentrations cannot be quantified would likely result in a significant underestimation of exposure for adsorbing substances.

Due to the good initial measured recoveries of the test substance as well as the indication of remaining test substance the extractions give it is acceptable to use measured initial values for adsorbing substances (See paragraph 40 OECD 2011). This approach was therefore used in preference to the use of Nominal or Geometric mean concentrations for calculation of the toxicological endpoints.

Results with reference substance (positive control):
The sensitivity of the algae was checked by performing a growth inhibition test with a reference compound (potassium dichromate) twice a year , and is not used unless found to be between the set EC50 values of 0.25 to 2.0 mg/L as indicated in the study guideline.
Reported statistics and error estimates:
Where possible, the EC10,20,50,80 values were computed from the best fitted line (least-squares method) through the points given by the probit of the percentage of inhibition and the logarithm of the concentration of the test substance. The EC50 value calculated for the area under the growth curve is termed EbC50, whereas the EC50 value calculated for the specific growth rate is termed ErC50. The Lowest Observed Effect Concentration (LOEC) was determined by comparison of the growth at each concentration and the control using threshold values from the Dunnetts test (Dunnetts, 1955). The No Observed Effect Concentration (NOEC) was derived from the results as the first concentration below the LOEC value, where growth shows no significant inhibition relative to the control values. All computations were performed using Toxcalc® 1994.

Due to the tendency of the substance to strongly adsorb additional replicates were added for complete extraction at T=0 in an attempt to improve the initial measured measurements.

Extraction from the algae cells and test vessels at the end of algae studies (T= 72) was conducted despite having limited reliability and reproducibility. Data was considered valuable however to demonstrate presence of test material at the end of the test and support use of measure initial concentrations as opposed to nominal or geometric mean measured concentrations

Validity criteria fulfilled:
yes
Conclusions:
Guideline study, GLP, All validity criteria fulfilled, the NaHCO3 concentration of the test medium was increased to 150 mg/L to maintain a more constant pH. This small change is not considered to have an influence on the test result. Idenfication of test substance complete, including chemical analyses. Low concentrations were observed at end of the test. Strong sorption of substance to algae. Mean measured initial concentration (±80% of nominal) are used for dose response according to OECD guideline.
Executive summary:

In order to predict the effects of the test chemical in an aquatic environment, the toxicity to freshwater algae was determined using the Algal Growth Inhibition Test in accordance with OECD, EC and ISO test guidelines and with the OECD Principles of Good Laboratory Practice. Slight modifications to the guideline were applied to ensure good growth and pH control of the cultures. The toxicity of the test substance to an exponentially growing culture of P. subcapitata was determined over an exposure period of 72 hours. All endpoints are based on measured initial concentrations. Significant effect (growth rate) was observed on growth rate at 0.04 mg/L. The NOEC is therefore <0.04 mg/L and LOEC is 0.04 mg/L. The ErC10 is 0.050 mg/L and the ErC50 is 0.23 mg/L.

The test is valid as shown by: The following quality criteria have been met in the present study:

· The cell density of the controls increased at least a factor 16 within 72 hours.

· The mean coefficient of variation for section-by-section specific growth rates in the control cultures must not exceed 35%.

· The coefficient of variation of average specific growth rates during the whole test period in the replicate control cultures should not exceed 7%.

· Analytical quality criteria were met.

· Algae strain used was of acceptable sensitivity.

In addition the pH variation in the control did not vary more than 0.6 pH units as recommended in the study guideline. The results of the chemical analyses show that the test substance concentration did not remain constant in the test system. The measured initial concentrations were all approximately 80% (79.9- 83.6%) of the nominal values. Due to the strongly adsorbing nature of the test substance use of measured initial concentrations is permitted in such cases by the test guideline.

Description of key information

One toxicity to quatic algae is available for tetramine di-C16-18. The ErC10 and ErC50 observed in this study are respectively 0.05 and 0.23 mg a.i./L based on measured initial concentrations.  

Key value for chemical safety assessment

EC50 for freshwater algae:
0.23 mg/L
EC10 or NOEC for freshwater algae:
0.05 mg/L

Additional information

There is one toxicity to aquatic algae test with tetramine di-C16 -18 available. The toxicity of tetramine di-C16 -18 to freshwater algae was determined using the Algal Growth Inhibition Test in accordance with OECD, EC and ISO test guidelines and with the OECD Principles of Good Laboratory Practice. Slight modifications to the guideline were applied to ensure good growth and pH control of the cultures. The toxicity of the test substance to an exponentially growing culture of P. subcapitata was determined over an exposure period of 72 hours. All endpoints are based on measured initial concentrations. Significant effect (growth rate) was observed on growth rate at 0.04 mg/L. The NOEC is therefore <0.04 mg/L and LOEC is 0.04 mg/L. The ErC10 is 0.050 mg/L and the ErC50 is 0.23 mg/L.

The results of the chemical analyses show that the test substance concentration did not remain constant in the test system. The measured initial concentrations were all approximately 80% (79.9- 83.6%) of the nominal values. Due to the strongly adsorbing nature of the test substance use of measured initial concentrations is permitted in such cases by the test guideline.