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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:
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
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
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:

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.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key 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:
The EC50 values, based on time-weighted mean measured test concentrations were greater than 1.4 mg/l and correspondingly the No Observed Effect Concentration was greater than or equal to 1.4 mg/l.
Executive summary:

The EC50 values, based on time-weighted mean measured test concentrations were greater than 1.4 mg/l and correspondingly the No Observed Effect Concentration was greater than or equal to 1.4 mg/l.

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 should 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

Two OECD 201 studies are available. In the first one conducted in 1997, no toxicity was observed up to the maximum achievable water solubility in the test (1.4 mg/L).

This test was repeated in 2013 with a better method of preparing the test substance solutions (the Water Accomodated Fraction), with a maximum loading rate of 514 mg/L. 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.