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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to protocol.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
bicarbonate levels in dilution medium increased, WAF was tested, endpoints based on loading rate
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
10 mL of all test concentrations were sampled for analysis at the start of the test and at the end of the test.
Vehicle:
no
Details on test solutions:
Preparation of solutions
A WAF method with a 1 day stirring time mimicking the conditions used in the solubility test was used for this study. This allowed both the maximum achievable concentration of parent and any degradation products generated in this period the possibility to dissolve up to their solubility limit in the test media. Thus assessing the direct effect the test substance a whole has on the test organism as well as the indirect effect of its more stable degradation products.
A traditional stock solution and subsequent dilutions were therefore not made during this test.
A WAF was prepared for each test concentration by adding of an accurate amount of the test substance to the test media and allowing equilibrate under slow stirring for 1 day. Each WAF was then considered loaded with the test substance components and / or breakdown products at their corresponding water solubility limits. The WAF solutions were then transferred to the test vessels as previously described.
The inocula were then added to each test vessel from an exponentially growing culture and the test vessels were stoppered and incubated for the test duration. In addition, 6 control replicates were tested containing test media only.
Due to higher concentrations being testing than in the range finding test significant amounts of un-dissolved test substance were still visible in the test vessels of the highest 2 concentrations with the chosen separation method in the first definitive study. A much higher than desired concentration was therefore present in these vessels. The separation technique used was therefore concluded inadequate. These invalid results are filed with raw data but not reported in further detail.
The lowest middle and highest concentrations were therefore repeated by amendment with identical procedures but with a fixed dispenser pipette so as to avoid repeatedly penetrating the surface layer where un-dissolved material was present.
Finally the middle WAF solution was made in duplicate and was itself inoculated and incubated under identical conditions with slow stirring for the purposes of gathering additional information.

Test concentrations
Using the WAF technique as previously detailed the following amounts of test substance were loaded to the test medium:
9.0, 92.2 and 944 mg/L.
The middle concentration was also tested directly as a WAF (in duplicate with a separate control) and was inccoculated separately without separation of the non dissolved material for the purpose of gathering additional data.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out with the freshwater unicellular algae P. subcapitata (CCAP 278/4) obtained from the Culture Collection of Algae and Protozoa, Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland, UK. After purchasing, this strain was cultured and maintained. Cultures on sloped agar tubes were stored at 4°C until required. Exponentially growing cultures are maintained at 23 ± 2°C in a temperature-controlled illuminated orbital incubator and are recultured under sterile conditions weekly to keep the algae in this phase. For the evaluation of the quality of the algae and the experimental conditions, the reference substance potassium dichromate was tested at least twice a year to demonstrate satisfactory test conditions and algae sensitivity
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.65 to 23.9 °C
pH:
7.9 to 9.9
Nominal and measured concentrations:
Nominal: 9.0, 92.2 and 944 mg/L
Measured: see attached tables.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL Erlenmeyer flasks with cotton wool stops.
- Type (delete if not applicable): open
- Aeration: no
- Initial cells density: 10 000 cells/mL
- Control end cells density: 2.1 x 10^6 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, OECD medium with 150 mg/L CaCO3 instead of 50 mg/L

TEST MEDIUM / WATER PARAMETERS
OECD medium with 150 mg/L CaCO3 instead of 50 mg/L
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 100.6 μmol/m2/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ~10
- Range finding study
- Results used to determine the conditions for the definitive study: Preliminary results showed slight effects at 100 mg/L loading. A full test was
therefore required. This result was sufficient to determine the concentration range for the definitive test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
92.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: endpoint based on loading rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 944 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: endpoint based on loading rate
Details on results:
The first definitive test contained visible un-dissolved test material in the highest 2 test concentrations. These results were therefore considered invalid by the study director and data was not used further. All raw data is filed for GLP purposes but no results have been calculated.
The second definitive test was conducted by amendment in an identical fashion but with an alternative separation technique as detailed in the methods section.
No un-dissolved material was visible during this test. These results are considered valid by the study director and have been used to express the
required endpoints.
The additional direct inoculation of the 92.2 mg/L WAF solution is also summarized in the results section. This is not a valid algae test and has not
been used for endpoint calculation but as additional information for the study director.

The test substance caused very minor inhibition of biomass and had a very slight effect on growth rate that was not detected as significant by the
statistical software using the Dunnetts test.
NOEC may then be expressed (for the test material as a whole) as 92.2 and EL50 as >944.1 mg/L. Alternatively when considering the parent chemical only it may be considered as non toxic to algae at its maximum achievable concentration in test medium.
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. The sensitivity was tested for compliance with the guidelines. The observed EC50 values were between 0.25 and 2.0 mg/L as required.
Reported statistics and error estimates:
Where possible, the EC10,50, 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. If this was not possible linear calculation methods were used. The LOEC was determined by comparison of the growth at each concentration and the control using the Williams or Dunnett’s tests. The 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 the TOXCALC V5.23.

Due to the speed the test chemical reaches saturation in water, preliminary information on half life and preliminary studies, a 1 day stirring period for the WAF vessels was chosen. This means that a mixture of the parent peroxide, degradation products (including temporary intermediate breakdown products) and any impurities were essentially tested for their effects on the test organism.

All critical validity criteria for the test and analytical method in the reported data were met. Data not meeting the required quality criteria is filed in the raw data file and has not been used further.

Chemical analysis of the parent only was conducted to confirm the presence and subsequent disappearance of the main peroxide ingredient. All concentrations measured when considering the error margins that can occur with such low measurements were essentially the same for a low middle or high loading concentration as would be expected with a poorly soluble test chemical.

Further measurement of all breakdown products was not in the scope of this study the focus being on the aquatic hazard of the product as a whole.

Results were expressed as loading values as is acceptable for test solutions containing multiple components. The concentration of the active ingredient was also measured as far as stability allowed at the start and end of the test.

To summarize, it is important to see the resulting test solution after 24 hours stirring as a complex mixture of parent material as well as breakdown products and any impurities that may be present. The soluble fraction of the test material generated from a loading of 92.2 mg/L can however be concluded as having no significant effect on the test organism. The directly inoculated WAF vessel did demonstrate more significant effect of the test organism. Due to this approach being a non guideline method these results were not used further but were conducted for a better understanding of the test system.

Validity criteria fulfilled:
yes
Conclusions:
This study is reliable. It is performed according to OECD guideline 201 under GLP and all validity criteria were fulfilled.
The test substance has a very low water solubiltiy and was stable during the test. Water Accommodated Fractions were created and endpoints are based on loading rates. The calculated endpoints are adequate for C&L and risk assessment purposes.
Executive summary:

In order to predict the effects of in an aquatic environment, an Algal Growth Inhibition test was conducted in accordance with OECD test guidelines and with the OECD Principles of Good Laboratory Practice. Some minor modifications to the guideline were applied due to inherent properties of the test chemical. These included preparing the test chemical as a water accommodated fraction (WAF), additional media components to aid pH stability and an extended stirring period before testing. An additional WAF based algae test with a single concentration and control was also conducted in parallel to the standard test to allow better understanding of test substance behavior.

The toxicity of the test chemical to an exponentially growing culture of P. subcapitata was determined over an exposure period of 72 hours. Nominal loading concentrations of 9.0, 92, and 944 mg/L were tested. For all concentrations no statistically significant effect in growth rate in comparison to the control was found.

The EL10 (72h) was calculated as >944.1 mg/L (Growth rate). The EL50 (72h) was estimated as >944 mg/L. (Growth rate).

The NOELR was determined as 92 mg/L (Growth rate) and the LOELR was determined as 944 mg/L (Growth rate).

Values are reported as loading rates (ELx values) which are equivalent to nominal concentrations for WAF preparations. Concentrations of active ingredient were measured at low, middle and high loading rates. Inhibition based on biomass has also been calculated should this be required by certain regulatory authorities.

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

- The EC50 value of the reference compound, potassium dichromate, was in the range of 0.25-2.0 mg/L (Documented as part of GLP laboratory maintenance).

- The mean coefficient of variation for section-by-section specific growth rates in the control did NOT exceed 35 %

The following criterion could not be met:

- Demonstration of the stability of all components of the test substance during the study. Due to the test substance degrading to multiple products, analytical monitoring of all of these was not feasible. Due to the low observed toxicity however this is not considered a critical limitation of the study. The parent component only was measured.

The study was considered an accurate representation of the effects the parent test chemical has at its maximum solubility limit in test media as well as demonstrating the lack of effect that any generated breakdown products may have had on the test organism.

Description of key information

One OECD 201 under GLP conditions has been performed for the substance. The test was prepared as Water Accomodated Fraction.  The EL10 (72h) was calculated as >944.1 mg/L (Growth rate). The EL50 (72h) was estimated as >944 mg/L. (Growth rate). The NOELR was determined as 92 mg/L (Growth rate) and the LOELR was determined as 944 mg/L (Growth rate). The results can also be expressed as the substance not being toxic to algae at it maximum water solubility limit.

Key value for chemical safety assessment

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

Additional information

In order to predict the effects of in an aquatic environment, an Algal Growth Inhibition test was conducted in accordance with OECD test guidelines and with the OECD Principles of Good Laboratory Practice. Some minor modifications to the guideline were applied due to inherent properties of the test chemical. These included preparing the test chemical as a water accommodated fraction (WAF), additional media components to aid pH stability and an extended stirring period before testing. An additional WAF based algae test with a single concentration and control was also conducted in parallel to the standard test to allow better understanding of test substance behavior.

The toxicity of the test chemical to an exponentially growing culture of P. subcapitata was determined over an exposure period of 72 hours. Nominal loading concentrations of 9.0, 92, and 944 mg/L were tested. For all concentrations no statistically significant effect in growth rate in comparison to the control was found.

The EL10 (72h) was calculated as >944.1 mg/L (Growth rate). The EL50 (72h) was estimated as >944 mg/L. (Growth rate).

The NOELR was determined as 92 mg/L (Growth rate) and the LOELR was determined as 944 mg/L (Growth rate).

Values are reported as loading rates (ELx values) which are equivalent to nominal concentrations for WAF preparations. Concentrations of active ingredient were measured at low, middle and high loading rates. Inhibition based on biomass has also been calculated should this be required by certain regulatory authorities.

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

- The EC50 value of the reference compound, potassium dichromate, was in the range of 0.25-2.0 mg/L (Documented as part of GLP laboratory maintenance).

- The mean coefficient of variation for section-by-section specific growth rates in the control did NOT exceed 35 %

The following criterion could not be met:

- Demonstration of the stability of all components of the test substance during the study. Due to the test substance degrading to multiple products, analytical monitoring of all of these was not feasible. Due to the low observed toxicity however this is not considered a critical limitation of the study. The parent component only was measured.

The study was considered an accurate representation of the effects the parent test chemical has at its maximum solubility limit in test media as well as demonstrating the lack of effect that any generated breakdown products may have had on the test organism.