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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:
23-1-2012-27-1-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study to appropriate guideline, with modifications relevant and justified for specific substance characteristics, critical quality criteria were met and endpoints were calculated with cconfidance limits.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
Medium adjusted to aid stability tested in sealed system
Principles of method if other than guideline:
Due to the rapid degradation of the test chemical to isobutyric acid (main degradation product) the buffer capacity of the test medium was increased
to assist pH stability. The test chemical was also observed to react with metal components of the test medium (during preliminary studies) resulting innutrient deficiency if not supplemented. 150% concentration of all the standard OECD medium ingredients was therefore used to ensure sufficient
growth.The halflife of the test chemical was such that degradation products were tested as these were considered most relevant for determining
aquatic hazard. A sealed system was used to ensure maximum possible exposure. A WAF (Water Accommodated Fraction) method was used as the
test substance itself is intentionally mixed with iso dodecane for stability / safety reasons and the active component degrades to multiple breakdown
products all of which could potentially contribute to toxicity. Separation and testing of individual components is therefore not possible.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No
Analytical monitoring:
yes
Details on sampling:
10 mL of all test concentrations were sampled for analysis at the start of the stirring period (after 15 minutes of rapid stirring) and at the end of the
stirring period (after 22 hours) and pipetted into sample pots on ice that were subsequently transferred to HPLC vials and analyzed for the active
peroxide component.
Vehicle:
no
Details on test solutions:
Due to the nature of the test chemical being unstable and mixed with iso-dodecane it requires testing in a manner that allows all of its
ingredients or resulting degradation products 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. A traditional stock
solution and subsequent dilutions were therefore not made during this test.

A WAF was prepared for each test concentration separately by adding of an accurate amount of the test substance to the test media and allowing
equilibrate under slow agitation over 24 hours in sealed vessels. Each WAF was then considered loaded with the test substance components and / or breakdown products at their corresponding water solubility limits. A GLP Hydrolysis test as well as in test analysis indicates that the main active ingredient of the test substance was no longer present after 24 hours . Erlenmeyer vessels were completely filled with the resulting WAF solutions (essentially degradation products, impurities in iso-dodecane) using a sterile dispenser. Each 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 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.
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.

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 100000 cells/mL in each of the test vessels.
Test type:
other: Static Sealed
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Cell obseved for bacterial contamination and morphological abnormalities only.
Hardness:
See other information for medium composition
Test temperature:
The temperature varied from 23.25 to 25.05 °C
pH:
Variation exceeded guideline norms. This is however easily explained by the lower starting pH used to offset a heavy pH decrease due to the acids
formed when the test chemical degrades. 7.1-10.3 in the control indicates good control growth and that the test organism is not effected by a slightly lower start pH.
Dissolved oxygen:
Not measured
Salinity:
See other information for medium composition
Nominal and measured concentrations:
Due to degradation products being tested measured initial concentrations are not relevant for expressing the study endpoints. Nominal or loading
concentrations were therefore used :

4.8, 15.6, 50.0, 160 and 512 mg/L were tested
Details on test conditions:
TEST SYSTEM
- Test vessel: 100ml Erlenmeyer
- Type: closed
- Material, size, headspace, fill volume: Completeley filled no headspace
- Control end cells density:10,000000 cells /ml
- No. of vessels per concentration (replicates):3
- No. of vessels per control (replicates):6


GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used: in other information

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Highest concentration was tested with and without pH adjustment to demonstrate an actual not pH related effect.
- Photoperiod:Continuous light
- Light intensity and quality:108.0 and 107.6 µmol•m-2•s-1 at the beginning and end of the test respectively


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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study : was conducted up to 100 mg/L and from this a definative range exceeding 100 mg/L was chosen due to lack of effect.
- Results used to determine the conditions for the definitive study: Preliminary studies showed removal of Iron from the OECD medium at higher
concentrations. The OECD medium was therefore compensated in the definative test to minimise effects due to unavailable micronutrients.
Previous non GLP research and a GLP hydrolysis study had identified the breakdown products and indicated the short halflife. The test design was
therefore chosen using this information and reccomendations in the "OECD 2000 GUIDANCE DOCUMENT ON AQUATIC TOXICITY TESTING OF
DIFFICULT SUBSTANCES AND MIXTURES. (OECD SERIES ON TESTING AND ASSESSMENT NUMBER 23"

Breakdown products include : Isobutyric acid, Isopropanol, propene and acetone
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
35 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 1.5-71.9 mg/L (95% CL)
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
151.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 75.1-313.3 (95 % CL)
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
15.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The test chemical did cause inhibition of algae growth at high concentrations. Dose response curves with confidence limits were produced. Due to the EL 50 based on growth rate being greater than 100 mg/L the test substance is not classifiable for its toxicity to algae species.
Results with reference substance (positive control):
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.
Reported statistics and error estimates:
The mean values of the observed absorbance for each test substance concencentration were used to calculate the growth and specific growth rate.
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. The LOEC was determined by comparison of the
growth at each concentration and the control using the William’s test. 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 compuations were performed using the TOCALC version V 5.0.23 . EC values can also be referred to as EL values when WAF preparations are
used. They are calculated in the same manner. In summary the maximum likelihood probit plot, the Williams test and the Shapiro-Wilks and the
Bartlett’s tests were used.

Medium Composition

Nutrient

Concentration

(mg/L)

Macro-nutrients

NH4Cl

22.5

KH2PO4

2.4

CaCl2(H2O)2

27

MgSO4(H2O)7

22.5

MgCl2(H2O)6

18.0

Fe-EDTA

FeCl3(H2O)6

0.096

Na2EDTA(H2O)2

0.15

Trace elements

H3BO3

0.277

ZnCl2

0.0045

MnCl2(H2O)4

0.622

CoCl2(H2O)6

0.0022

CuCl2(H2O)2

0.15x10-5

Na2MoO4(H2O)2

0.0105

NaHCO3

NaHCO3

150 [Modified]

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid.

200 (HEPES buffer) [Modified]

Results Table

Concentration

ABSORBANCE

 

Time (hours)

 

AUC

Inhibition

%

SGR

Inhibition

%

(mg/L)

0

24

45

72

 

 

 

 

0.000

0.011

0.059

0.350

0.603

16.392

 

0.057

 

0.000

0.009

0.026

0.290

0.574

13.932

 

0.062

 

0.000

0.008

0.044

0.274

0.556

13.824

 

0.061

 

0.000

0.009

0.032

0.291

0.599

14.400

 

0.062

 

0.000

0.007

0.033

0.271

0.619

14.304

 

0.065

 

0.000

0.009

0.026

0.303

0.646

15.108

 

0.064

 

Mean

0.009

0.037

0.297

0.600

14.660

 0.0

0.061

0.0

 

 

 

 

 

 

 

 

 

4.800

0.009

0.043

0.272

0.583

14.016

 

0.060

 

4.800

0.010

0.046

0.312

0.675

16.092

 

0.061

 

4.800

0.008

0.041

0.266

0.601

14.100

 

0.062

 

Mean

0.009

0.043

0.283

0.620

14.736

-0.5

0.061

0.8

 

 

 

 

 

 

 

 

 

15.600

0.008

0.035

0.264

0.664

14.664

 

0.063

 

15.600

0.009

0.044

0.261

0.707

15.264

 

0.061

 

15.600

0.008

0.040

0.255

0.674

14.688

 

0.062

 

Mean

0.008

0.040

0.260

0.682

14.872

-1.4

0.062

-1.1

 

 

 

 

 

 

 

 

 

50.000

0.008

0.042

0.203

0.488

11.256

 

0.056

 

50.000

0.008

0.044

0.224

0.582

12.936

 

0.059

 

50.000

0.007

0.044

0.223

0.511

12.120

 

0.058

 

Mean

0.008

0.043

0.217

0.527

12.104

17.4

0.058

6.0

 

 

 

 

 

 

 

 

 

160.000

0.008

0.029

0.041

0.041

1.692

 

0.021

 

160.000

0.008

0.019

0.034

0.043

1.308

 

0.022

 

160.000

0.008

0.025

0.035

0.038

1.416

 

0.020

 

Mean

0.008

0.024

0.037

0.041

1.472

90.0

0.021

65.8

 

 

 

 

 

 

 

 

 

512.000

0.002

0.015

0.021

0.022

1.008

 

0.017

 

512.000

0.004

0.010

0.021

0.020

0.744

 

0.014

 

512.000

0.003

0.015

0.027

0.028

1.164

 

0.019

 

Mean

0.003

0.013

0.023

0.023

0.972

93.4

0.017

72.9

AUC= Area under curve (Biomass)

SGR = Specific Growth Rate (Rate)

Validity criteria fulfilled:
yes
Conclusions:
The study can be considered reliable without significant restriction.
Executive summary:

A guideline study to GLP with supplementary chemical analysis and satisfactory substance identification was carried out with justified deviations from the guideline due to the inherent characteristics of the tests substance. All critical validity criteria were met and the study can be considered a reliable worst case representation of the effects the test substance to P.Subcapitata. Due to the relatively low toxicity observed (EL50 >100 mg/L) and the ready biodegradability of the test substance and it breakdown products the fact that the breakdown products were not separately quantified is not considered a critical flaw to the study.

Description of key information

Due to the extremely short half-life of the test material determined in the hydrolysis study the degradation products of the test material were deemed the most relevant for aquatic toxicity. For this reason the test material was loaded to the test medium agitated gently for 24 hours and then used directly for testing. All test concentrations were prepared separately as Water Accommodated Fractions to allow the determination of the toxicity of the resulting degradation mixture as a whole to be determined. It is known that the test chemical degrades to multiple degradation products and is itself present in isododecane solvent. The effects cannot therefore be attributed to a single component with 100% certainty however the main degradation product isobutryic acid is the most likely cause of the slight toxicity observed.  

ECHA data on Isobutyric acid gives 72h-EC50 values of 45.1 mg/L (biomass) and 44.7 mg/L (fluorescence) (ECHA dossier 79 -31 -2)

.

Key value for chemical safety assessment

EC50 for freshwater algae:
151.2 mg/L
EC10 or NOEC for freshwater algae:
35 mg/L

Additional information

Due to the extremely short half-life of the test material determined in the hydrolysis study the degradation products of the test material were deemed the most relevant for aquatic toxicity. For this reason the test material was loaded to the test medium agitated gently for 24 hours and then used directly for testing. Effects to algae were observed and an EL10 and an EL50 were determined. The test material is not of high concern to algae species as the EL50 was greater than 100 mg/L of test material and would therefore not be classified for this endpoint. For the algae endpoint exact EL50 values were determined. For this reason the algae endpoint was used for PNEC derivation.