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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:
Jan 2015 - May 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study done under GLP, well documented
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples were taken from all concentrations and the control at the start and at the end of the test. At the end of the test extra samples from parallel replicates without algae were also taken. At least 15 mL was sampled in each case. Samples from the actual test replicates 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 and samples were then added to HPLC vials. The samples were analyzed as soon as possible after sampling no storage took place.
Vehicle:
no
Details on test solutions:
Test solutions
Preparation of the test solutions
The test material comprises of multiple components with different physical chemical properties. The active components are also of low solubility (AkzoNobel 2015). For this reason preparation of the test solutions in a standard manner via a stock solution and further dilution is not possible. For this reason WAF’s were prepared for each test concentration separately. This was achieved by accurate weighing of the test substance with an analytical balance and loading of the test material to 1 liter of test medium. The resulting solutions were stirred slowly for approximately 70 hours. This was shown by existing water solubility studies as sufficient to achieve the maximum solubility of the measured active components. After stirring, the solutions were stopped and allowed to rest for one hour. After which approximately 300 ml of liquid from each of the WAF vessels was removed with a dispenser pump positioned in the middle of the water phase so as to avoiding transfer of undissolved material on the surface or bottom of the vessels. Centrifugation then took place at 8000 RPM for 10 minutes. This also reduces the chance of undissolved material in the form of a dispersion being transferred to the test. From the centrifuge vessels the test vessels were filled to 40 ml with the use of a dispenser pipette. Prior to each concentration the pipette was primed with the corresponding WAF solution that was then discarded to reduce any possible adsorption of test substance to the apparatus. The test solutions were then ready for addition of test organisms and absorbance / physical chemical measurements.
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 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.

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 extinction of an exponentially growing stock culture was measured. The cell density was determined using the calibration curve described in section 3.5. From this algal culture a dilution was
prepared to obtain an initial cell density of approximately 1×104 cells/mL in the test medium
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21.8 to 22.2 °C
pH:
7.7 - 8.5
Nominal and measured concentrations:
The following final nominal test substance loadings were prepared: 8.1, 23.9, 66.3, 203.5 and 608.5 mg/L.
Details on test conditions:
Test procedures
Culture medium was prepared by diluting the stock mineral salts in an appropriate vessel. This medium was sterilized by filter sterilization (0.2 μm). Adequate amounts of test substance stock solution were added. The Erlenmeyer flasks were then filled with test solution up to a total volume of 40 mL using a sterilized dispenser pipette. Then the inoculum was added to the vessel from an exponentially growing culture. All test concentrations were tested in triplicate. In addition, six replicates of the control were included.
The extinction of light in each Erlenmeyer flask was measured after 0, 24, 48 and 72 hours. Algal medium was used as a blank in the spectrophotometer.
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
18.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 608.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
5.52 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
43 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass

Analytical results

The concentration of the test substance present in the test vessels was quantified using the method as described in Annex 7 and the results are presented in Table I and II in section 8.The method used met all validity requirements as can be seen in Table III. The concentrations measured during the test were not stable within 80 to 120% of the initial concentrations. Therefore, geometric measured mean concentrations of the filtered (in test) solutions were calculated despite their limited use in a complex mixture. Where recovery was <LOQ, ½ of the quantification limit was used. Measurement of the recovery in the test vessels was sufficient and the data generated in the parallel replicates was not used further as it was not required. The measured initial concentrations demonstrate that when the test substance is loaded at a concentration greater than the solubility of a particular component, that only marginal or no further increase in the initial concentration of that component occurs. This indicates that the maximum achievable concentrations of the measured components were reached at the start of the test

Validity criteria fulfilled:
yes
Conclusions:
The ELC10 (rate) is 18.6 mg/L. The ELC50 (rate) was not reached at the highest concentration and can therefore only be reported as >608.5 mg/L.
Executive summary:

In order to estimate the effects of the test chemical in an aquatic environment, the toxicity of the soluble components of the test substance 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 and appropriate testing of a complex mixture.

The toxicity of the test substance to an exponentially growing culture of P. subcapitata was determined over an exposure period of 72 hours. Nominal loadings of 8.1, 23.9, 66.3, 203.5 and 608.5 mg/L were tested including the required control group. Significant effect was observed on growth rate at 23.9 mg/L. The NOELR (rate) is therefore 8.1 mg/L and LOELR is 23.9 mg/L. The ELC10 (rate) is 18.6 mg/L. The ELC50 (rate) was not reached at the highest concentration and can therefore only be reported as >608.5 mg/L.

Significant effect was observed on growth at 23.9 mg/L. The NOELR (biomass) is therefore 8.1 mg/L and LOELR is 23.9 mg/L. The ELC10 (biomass) is 5.52 mg/L. The ELC50 (biomass) was found to be 43.0 mg/L.

The test is valid as shown by: The increase of the extinction of the control over 72 h by a factor of 180. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35%. The coefficient of variation of average specific growth rates during the whole test period in the replicate control cultures did not exceed 7%. The set quality criteria for chemical analysis were all met. The most recent reference test for the algae species tested demonstrated acceptable sensitivity according to the test guideline. The following quality criterion was not met in the present study:

The results of the chemical analyses show that the measured components did not remain stable in the test system. However due to the substance being a complex mixture, nominal loadings of test material were the most appropriate way to express the toxicity.

pH variation in the control did not vary more than 0.8 pH units as recommended in the study guideline.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
19/2/2013-22/2/2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Appropriate guideline followed with modifications for poorly soluble substances. No chemical analysis or GLP accreditation. Restricted for use as supporting evidence only.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Principles of method if other than guideline:
Less replicates used, no chemical analysis, WAF method was used. Study is for screening purposes only.
GLP compliance:
no
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
N/A
Analytical monitoring:
no
Details on sampling:
N/A
Vehicle:
no
Details on test solutions:
The medium, described by OECD guideline 201 was used without modification as the test media.

The test chemical is mixed with isoparaffinic hydrocarbons for stability and safety reasons and cannot be tested separately. The test material therefore needs to be tested in a manner that allows all of its ingredients or resulting degradation products / impurities the possibility to dissolve up to their solubility limit in the test media and thus assessing ecotoxicological effect of all of the components of the test chemical. For this reason a WAF approach was used for this test. WAF solutions at 1,10 and 100 mg/L were prepared as follows; accurately weighed amounts of test material (1 10 and 100 mg) were added to 1000 mL of OECD algae medium was added and agitated slowly with a magnetic stirrer at room temperature for 24 hours in a sealed glass vessel. The procedure was repeated for each WAF solution separately. After 24 hours each WAF was then considered loaded with the test substance.

Each WAF was left to stand for one hour after which the test solutions were extracted from the middle of the test vessels using a syringe and inert tubing. Due to the possibility of transferring un-dissolved test material the removed solutions were centrifuged at 8000 RPM for 10 minutes and then transferred in the same manner to the test vessels. Each WAF was prepaired separately and each WAF solution was used directly as the test concentration after centrifugation.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Pseudokirchneriella subcapitata no other data.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Observed for bacterial contamination only.
Hardness:
Not measured
Test temperature:
Incubators set at 23 ºC. Varied <2 ºC during the study.
pH:
8.0 at the start of the test
Dissolved oxygen:
Not measured
Salinity:
Not measured
Nominal and measured concentrations:
Standard algae screening test concentrations of 1.0, 10 and 100 mg/l were prepared as WAF’s.
Details on test conditions:
For the tests adequate amounts of OECD algae test medium were prepared in an appropriately sized volumetric vessel. This medium was sterilized by filter sterilization (0.45µm filter). This was used to create the WAF solutions as detailed in test solutions. 100 ml Erlenmeyer’s were used with 40 ml total volume. After WAF generation the inoculum was added from an exponentially growing culture with a pipette. Each test concentration was tested in triplicate and 4 replicates of the control without test substances were included. The extinction in each Erlenmeyer was measured after 0 and 72 hours. Algal medium was used as a blank in the spectrophotometer to correct for media absorbance. Light intensity was set at 100 µmol.m-2 .s-1.

No chemical analysis for quantification of the test substance concentration was performed during the studies.
Reference substance (positive control):
yes
Remarks:
Conducted as part of laboratory GLP maintainence.
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: N/A
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 10 - < 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: N/A
Details on results:
Growth rate inhibition was observed at the highest concentration.
Reported statistics and error estimates:
Not conducted (Screening Study)
Validity criteria fulfilled:
yes
Remarks:
Valid as supporting evidence only.
Conclusions:
The test results are valid in support of the key study. Control performance sufficient OECD guideline followed.
Executive summary:

Non GLP screening study to relevant guideline without chemical analysis and with adaptions for poorly water soluble substances. Valid as supporting evidence with the restrictions mentioned.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
17-6-2013 - 21-6-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study with justified deviations from a relevant guideline. Chemical analysis and Certificate of analysis present. Critical validity criteria met. considered a reliable representation of effects to algae without restriction.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
Elevated Sodium Hydrogen Bicarbonate + Elevated Iron content in medium
Principles of method if other than guideline:
Principles of guideline followed but adapted for WAF (water accommodated fraction) preparations as detailed in the OECD series on testing and assessment number 23. (Guidance document on aquatic testing of difficult substances and mixtures)
GLP compliance:
yes
Remarks:
Awaiting report inspection (Study plan and test inspected)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
N/A
Analytical monitoring:
yes
Details on sampling:
25 mL of each test concentration was sampled for analysis at the start of the test (after stirring period of 1 day) and at the end of the test 30 ml (3x10ml pooled sample) were taken at each test concentration and. In parallel vessels (without test organisms) a single 30 ml sample was taken per concentration. All of the samples taken were analyzed. No leaching solution or stabilizing agent was used. Samples were taken and analyzed directly in test medium.
Vehicle:
no
Details on test solutions:
Preparation of solutions

Due to the test substance being a mixture, of low solubility and potentially instable an appropriate preparation method was required.

A WAF method with a 1 day stirring time 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.

A traditional stock solution and subsequent dilutions were therefore not made during this test.

Culture medium was prepared by diluting the OECD stock mineral salts in an appropriate vessel with de-ionized water. The medium was then sterilized by filter sterilization. The WAF solutions at the desired test concentrations were then prepared separately by accurate weighing of test substance
for each WAF separately. Vessels were then sealed and left to stir slowly for approximately 24 hours and then allowed stand for approximately 1 hour. After which each solution was individually siphoned into a centrifuge tube and centrifuged at 8000 RPM for 10 minutes at 20ºC. The resulting solutions were transferred to the test vessels from the middle of the centrifuge tube avoiding as far as possible the transfer of surface film and/or un-dissolved test substance. The test vessels were then inoculated with the test organism and sealed with a cotton wool stopper and incubated for 72 hours. Absorbance was measured spectrophotometrically at 436 nm after 0, 24, 48 and 72 hours to allow determination of the desired endpoints. Due to the tendency of some peroxides to adhere to test apparatus (potentially reducing exposure) all apparatus including centrifuge tubes were
rinsed with the corresponding test solution prior to use to minimize adhesion to test apparatus.

The inoculum was then added to each test vessel from an exponentially growing culture and the test vessel was sealed and incubated for the test d
uration. 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.








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 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
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Examined for bacterial contamination only.
Hardness:
Not measured (Modified OECD medium used)
Test temperature:
22.2 - 22.9 °C
pH:
8.1-10.3 (variation in control)
Dissolved oxygen:
N/A
Salinity:
Not measured (Modified OECD medium used)
Nominal and measured concentrations:
5.0, 16.2, 51.2, 163.4 and 524.3 mg/L. (Nominal Loading) . See analytical results for measured concentrations.
Details on test conditions:


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

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

General test principles and procedures
Culture medium was prepared by diluting the OECD stock mineral salts in an appropriate vessel with de-ionized water. The medium was then sterilized by filter sterilization. The WAF solutions at the desired test concentrations were then prepared separately, sealed and left to stir slowly for approximately 24 hours and then stand for approximately 1 hour. After which each solution was individually siphoned into a centrifuge tube and centrifuged at 8000 RPM for 10 minutes at 20ºC. The resulting solutions were transferred to the test vessels from the middle of the centrifuge tube avoiding as far as possible the transfer of surface film and/or un-dissolved test substance. The test vessels were then inoculated with the test organism and sealed with a cotton wool stopper and incubated for 72 hours. Absorbance was measured spectrophotometrically at 436 nm after 0, 24, 48 and 72 hours to allow determination of the desired endpoints. Due to the tendency of some peroxides to adhere to test apparatus all apparatus including centrifuge tubes were rinsed with the corresponding test solution prior to use to minimize adhesion to test apparatus. Test concentrations
were tested in triplicate and the control was conducted with 6 test vessels.
Reference substance (positive control):
yes
Remarks:
Conducted as part of reoutine laboratory maintainence
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
44 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: No CL calculated
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
115.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
(No EL50 for Rate could be reliably calculated)
Remarks on result:
other: CL Calculated
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 15.7 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: Maximum achievable concentration at start of test in test medium
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 4.32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: Geometric mean of start and end concentrations (starting with test substance at maximum achievable solubility)
Details on results:
Note: quantification was conducted by the measurement of the active ingredient but this was related back to total amount of substance via a calibration curve. Measured values are not measured active as such but total product quantified by measurment of the active ingredient. The test substance can be considered as non toxic to algae. Both for chronic EC10 and EC50 endpoints. The EC50 value displayed here is derived from the biomass results as the test material was not sufficiently toxic to cause 50% rate inhibition at a loading concentration of 524.3 mg/L. Due to the relitively low toxicity of the solvent added to this substance for stability it may be desirable to express toxicity as the active component only. In whichcase the measured data demonstrates that the EC50 is in excess of the maximum achievable concentration of the active component in the test medium. The conclusion shoud therefore be no acute or chronic toxicity to algae at the limit of solubility for this test substance.
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:
Dunetts test was used for the detection of significant difference between the contol and treatments. A maximum liklehood probit plot was used for determination of the ECx values. All calculations were caried out using Toxcalc (Tidepool Scientific).

Analytical Results

Sample

Concentration (mg/L)

 

T=0h

T=72h

Geo mean

T=72h

(parallel)

Control

< LOQ

< LOQ

-

< LOQ

5 mg/L

3.1

0.06

0.43

0.05

16.2 mg/L

9.6

0.34

1.8

0.51

51.2 mg/L

13.4

0.74

3.14

1.07

163.4 mg/L

15.6

0.25

1.97

0.94

524.3 mg/L

15.7

1.19

4.32

0.26

Test Medium

Nutrient

Concentration

(mg/L)

Macro-nutrients

NH4Cl

15

KH2PO4

1.6

CaCl2(H2O)2

18

MgSO4(H2O)7

15

MgCl2(H2O)6

12

Fe-EDTA

FeCl3(H2O)6

0.096 (Elevated)

Na2EDTA(H2O)2

0.15   (Elevated)

Trace elements

H3BO3

0.185

ZnCl2

0.003

MnCl2(H2O)4

0.415

CoCl2(H2O)6

0.0015

CuCl2(H2O)2

1x10-5

Na2MoO4(H2O)2

0.007

NaHCO3

NaHCO3

150 (Elevated)

Validity criteria fulfilled:
yes
Remarks:
Yes
Conclusions:
This test may be considered reliable without restriction. When considering all measured algae endpoints (including supporting data) the test material may be concluded non toxic to algae at its maximum achievable concentration in test medium.
Executive summary:

GLP study with justified deviations from a relevant guideline. Chemical analysis and Certificate of analysis present. Critical validity criteria met. Can be considered a reliable representation of effects to algae without restriction.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
10-1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was performed under GLP and is considered complete
Qualifier:
according to
Guideline:
other: OECD 201 and C3 of EU guideline 92/69/EEC
GLP compliance:
yes
Analytical monitoring:
yes
Vehicle:
yes
Details on test solutions:
Identity and concentration of auxiliary solvent for dispersal: Acetone
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
CA 100 mg CaCO3/L
pH:
7.6-9.7
Nominal and measured concentrations:
nominal 2.0 mg/L
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.4 mg/L
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.4 mg/L
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 1.4 mg/L
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 1.4 mg/L
Basis for effect:
biomass
Details on results:
%Concentration loss over test: ... 36
Validity criteria fulfilled:
yes
Conclusions:
study is valid and complete

Description of key information

The results of a valid OECD 201 test indicate based on measured data that the EC50 is in excess of the maximum achievable concentration of the active component in the test medium. The conclusion shoud therefore be no acute or chronic toxicity to algae at the limit of solubility for this test substance.

Key value for chemical safety assessment

Additional information

In order to estimate the effects of the test chemical in an aquatic environment, the toxicity of the soluble components of the test substance 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.

The toxicity of the test substance to an exponentially growing culture of P. subcapitata was determined over an exposure period of 72 hours. Nominal loadings of 8.1, 23.9, 66.3, 203.5 and 608.5 mg/L were tested including the required control group. Significant effect was observed on growth rate at 23.9 mg/L. The NOELR (rate) is therefore 8.1 mg/L and LOELR is 23.9 mg/L. The ELC10 (rate) is 18.6 mg/L. The ELC50 (rate) was not reached at the highest concentration and can therefore only be reported as >608.5 mg/L.

Significant effect was observed on growth at 23.9 mg/L. The NOELR (biomass) is therefore 8.1 mg/L and LOELR is 23.9 mg/L. The ELC10 (biomass) is 5.52 mg/L. The ELC50 (biomass) was found to be 43.0 mg/L.

The test is valid as shown by: The increase of the extinction of the control over 72 h by a factor of 180. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35%. The coefficient of variation of average specific growth rates during the whole test period in the replicate control cultures did not exceed 7%. The set quality criteria for chemical analysis were all met. The most recent reference test for the algae species tested demonstrated acceptable sensitivity according to the test guideline. The following quality criterion was not met in the present study:

The results of the chemical analyses show that the measured components did not remain stable in the test system. However due to the substance being a complex mixture, nominal loadings of test material were the most appropriate way to express the toxicity.

pH variation in the control did not vary more than 0.8 pH units as recommended in the study guideline.