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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:
May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD 201 guideline study under GLP with analytical monitoring
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
Increased levels of NaHCO3 (150mg/L) were added to the test medium to stabilise the pH
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
General test principles and procedures
Culture medium was prepared by diluting the OECD stock mineral salts as indicated in Table 1 in an appropriate vessel with de-ionized water. The medium was then sterilized by filter sterilization.

The nature of the test chemical being very poorly soluble potentially hydrolytically and thermically instable require the substance to be tested in a manner that allows all of its ingredients or resulting degradation / hydrolysis products the possibility to dissolve up to their solubility limit in the test media. Therefore assessing ecotoxicological effect of all possible components in or originating from the original test chemical. For this reason a WAF approach was chosen for this test. A traditional stock solution and subsequent dilutions were not made and use of high energy dispersion or solvent was intentionally avoided.

A WAF (Water Accommodated Fraction) solution at the desired test concentration were prepared and used directly as a test solution after the stirring period. A control prepared in an identical manner without test substance was also prepared. Measurements made at 0, 24, 48 and 72 hours were used for effect determination by comparison to the control with an appropriate statistical method. In addition the WAF vessel was also inoculated and incubated in the same manner as normal test vessels.

Preparation of solutions
A WAF was prepared for the test concentration by adding of an accurate amount of the test substance weighed on an analytical balance vastly in excess of the water solubility limit to the test media. This solution was allowed to equilibrate under slow-moderate agitation over 24 hours in sealed 1 L glass vessels and then stand for approximately an hour before use. Each WAF was then considered loaded with the test substance components and / or breakdown products at their corresponding water solubility limits and was transferred without solid test material or film to the test vessels. The test vessels were filled with 40 mL of the corresponding WAF test solution or control. The test concentration was tested in triplicate prepared from the same WAF. The inoculum was then added to the vessel from an exponentially growing culture and the test vessel was sealed with a cotton wool stopper and incubated for the test duration. In addition, 6 control replicates were tested containing test media only. The extinction of the contents of each Erlenmeyer flask was measured after 0, 24, 48 and 72 hours. The remaining 100mg/L WAF solution and an additionally prepared 100 mg/L WAF were also directly inoculated and incubated as is without removal of the soluble fraction and were otherwise handled in the same manner as normal test vessels.

Test concentrations
2 x 1 L WAF loaded with a 100 mg of test substance and 2 x 1 L control containing only test medium were made as previously described and water accommodated fraction was used directly for testing. The remaining WAF solutions (and accompanying controls) were also inoculated and tested in the same manner to establish any influence the un-dissolved test chemical may have on algae growth.



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 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 re-cultured 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

Preparation of the inoculum
The initial stock culture was inoculated with P. subcapitata from a sloped agar tube and checked for purity by microscopic means. This algal stock culture (40 mL) of P. subcapitata was regularly transferred to fresh medium to act as inoculum for testing. The absorbance of an exponentially growing stock culture was measured. The cell density was determined using the calibration curve described below. From this algal culture a dilution was prepared to obtain an initial cell density of approximately 1104 cells/mL in each of the test vessels.


Quality control of the algae
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 should be between 0.25 and 2.0 mg/L.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
CaCl2(H2O)2 18 mg/L
MgSO4(H2O)7 15 mg/L
MgCl2(H2O)6 12 mg/L
Test temperature:
22.2 - 22.9 oC
pH:
8.0 - 9.3
Dissolved oxygen:
not relevant
Nominal and measured concentrations:
The following amounts of test substance were loaded to the test medium: 8.0, 25.6, 81.9, 262.1 and 838.4 mg/L.
Details on test conditions:
Chemicals
All reagents used were of reagent grade quality.

De-ionized water The de-ionized water used contained not more than 0.01 mg/L of copper, had a TOC-content of not more than 2.0 mg/L and a conductivity of less than 5 S/cm. This water was produced from tap water in a water purification system according to the relevant Standard Operation Procedure.

Test flasks
The test was performed in sterile 100 mL Erlenmeyer flasks with cotton wool stoppers.

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. 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. The test vessels were agitated continuously at a speed sufficient to prevent sedimentation of the algae (100 rpm approx).

Other Apparatus
The pH was determined with a microcomputer pH meter. The temperature was measured with a temperature sensor and data recorder. The light intensity was measured with a light intensity meter. The algae purity was inspected using a microscope.

Determination of pH, temperature, light, and purity of algae
The pH of all samples and controls were measured at the beginning and at the end of the test using a pH meter. The temperature in the culturing apparatus was continuously measured by a temperature sensor and data recorder which was read out at the end of the test. The light intensity was measured at the beginning and at the end of the test using a light intensity meter. At the end of the test five random samples were microscopically checked for purity of the algal culture.
Reference substance (positive control):
yes
Remarks:
potassium dchromate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Results with reference substance (positive control):
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).
Reported statistics and error estimates:
Evaluation of data was conducted using the Toxcalc 5.0 software package and Wilcoxons two sample test to check for significant difference in comparison to the control (Ref 3).

Preliminary tests

A preliminary test was conducted using identical methods and materials as detailed for the definitive test. No toxic effects on the test organisms were observed.

Definitive test

The guideline quality criteria for the coefficients of variation for average specific growth rates during the whole test period in the control and the mean coefficient of variation for section by section specific growth rates in the control are displayed in Table 3. Statistical comparison of the test replicate with the control indicated a NOELR of 100 mg/L and a LOELR of >100 mg/L. The EL50 is therefore also > 100 mg/L. The test chemical may also be considered as non toxic at its solubility limit in test medium in a standard algae test. Additionally no visible effects were seen in the additional inoculated WAF vessel.

pH, temperature, light and purity of algae

The pH measurements showed a maximum increase of 1.7 pH units in the control replicates which exceeds the guideline recommendation of 1.5 slightly. This is however easily explained by a very good control growth that slightly higher than usually achieved in 72 hours. The temperature varied from 21.95 to 22.7 °C during the test, and the light intensity was 115.2 and 114.8 µmol·m-2·s-1 at the beginning and end of the test respectively. Light and temperature measurements were in accordance with the required conditions described in the study plan. 2 control cultures and 3 test replicates were inspected microscopically at the end of the test and no significant bacterial contamination was observed.

Validity criteria fulfilled:
yes
Remarks:
cell density of the controls increased at least a factor 16 within 72 h; coefficient of variation of average specific growth rates during the whole test period in the replicate control cultures did not exceed 7% ;
Conclusions:
Under the conditions of the test, the EL10 (72h) >100 mg/L of test material.
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), minor changes in media components to help aid pH stability.

The toxicity of the test chemical to an exponentially growing culture of P. subcapitata was determined over an exposure period of 72 hours. A nominal loading concentration of 100 mg/L as a limit test was conducted. In addition to the testing of the water accommodated fraction in the standard manner (i.e. without un-dissolved material), the WAF vessel itself including any solid undissolved test chemical was also inoculated with algae and growth was monitored to allow additional data to be obtained.

No statistical difference in growth rate or biomass was detected in comparison to the control. The test substance was concluded as not toxic to algae at its maximum achievable solubility limit in the test medium.

NOELR >100 mg/L of test material.

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 % (See Table 3).

The following criteria could not be determined:

  • The stability of the test chemical during the test period.

Chemical analysis was not possible due to the extremely low solubility of the test chemical. This study was therefore conducted without chemical analysis. Additional efforts were therefore made to obtain data form a realistic worst case scenario as well as a standard test setup so as to allow a sufficient assessment of any hazard to fresh water algae in the absence of analytics.

Description of key information

One OECD 201 study is available with The EL10 (72h, growth rate) > 100 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L

Additional information

One OECD 201 study carried out under GLP is available.

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), minor changes in media components to help aid pH stability.

The toxicity of the test chemical to an exponentially growing culture of P. subcapitata was determined over an exposure period of 72 hours. A nominal loading concentration of 100 mg/L as a limit test was conducted. In addition to the testing of the water accommodated fraction in the standard manner (i.e. without un-dissolved material), the WAF vessel itself including any solid undissolved test chemical was also inoculated with algae and growth was monitored to allow additional data to be obtained.

No statistical difference in growth rate or biomass was detected in comparison to the control. The test substance was concluded as not toxic to algae at its maximum achievable solubility limit in the test medium.

NOELR >100 mg/L of test material.