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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to the category approach justification provided in section 13.
Reason / purpose:
read-across source
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
Source, key, 5274-61-3, BASF, 1997, R9700267, D. subspicatus, 72 h, RL2
Duration:
72 h
Dose descriptor:
EC0
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other:
Remarks:
Source, key, 5274-61-3, BASF, 1997, R9700267, D. subspicatus, 72 h, RL2
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading of a water accommodated fraction
Remarks on result:
other:
Remarks:
Source, WoE, 68439-49-6, BASF, 2015, 60E0461/14X174, P. subcapitata, 72 h, RL2
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate of a water accommodated fraction
Remarks on result:
other:
Remarks:
Source, WoE, 68439-49-6, BASF, 2015, 60E0461/14X174, P. subcapitata, 72 h, RL2
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
24 - 27 Mar 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study but lack of data and incomplete information
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
not specified
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: a 100 mg test item/L stock solution was prepared directly in the mineral medium. Solution was stirred 24 h and insoluble phase was subsequently filtered through a glass fiber filter. The filter was pre-treated with NaOH and again neutralized. Aliquots of this stock solution were filled in the test vessels for the actual test. The algae suspension was thereafter added to the test vessels.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Source (laboratory, culture collection): Pflanzenphysiologisches Institut der Universität Göttingen, supplied on 21.11.1996, a culture is maintained for 6 months in the laboratories of Henkel KGaA

ACCLIMATION
- Acclimation period: 3-4 days pre-incubation in test medium without test item
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
23.0 - 23.1 °C
pH:
7.9 - 8.4
Nominal and measured concentrations:
Nominal test concentration: 100 mg/L
Measured concentration: below detection limit (0.1 mg/L)
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks
- Fill volume: 100 mL
- Initial cells density: 10E+04 cells/mL
- Control end cells density: 3.7E+05 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: according to EU-guideline 92/69/EWG

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- Light intensity and quality: 2000 Lux (daylight-spectrum)

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: cell numbers were determined after 24, 48, 72 h

TEST CONCENTRATIONS
- Range finding study: yes, (10 - 14 Mar 1997, no further data available)
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC0
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: the test was conducted with a concentration of the test substance laying over the solubility limit. The insoluble phase was filtered prior to test initiation. The test substance recovery by analytical methods was under the detection limit of 0.1 mg/L. Therefore results refer to nominal values.

The test substance was not toxic to aquatic algae up to the limit of water solubility.

EyC50 (72 h) was > 100 mg/L.

Table 1: Cell concentration at different incubation timepoints

Substance concentration [mg/L] Algae cell concentration [cells/mL]
24 h 48 h 72 h
0 3.40E+04 7.80E+04 3.70E+05
100 3.30E+04 7.40E+04 3.60E+05
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
27 Nov 2014 - 05 Feb 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Arbeit und Soziales des Landes Nordrhein-Westfalen
Analytical monitoring:
yes
Details on sampling:
- Sampling method: 200 mL samples were taken from the replicates, given in glass vials (the test item concentrations were analysed in all test solutions sampled at the beginning of the exposure period prior to addition of the algae and in samples of the algae cultures taken from representative replicates per test concentration at the end of the exposure period)
- Sample storage conditions before analysis: Analysed immediately, additinal aliquots of the samples were stored as retain samples at -20 °C.
Vehicle:
no
Details on test solutions:
Due to the very low water solubility of the test item, the test media were individually prepared by applying the Water Accommodated Fraction (WAF) approach according to the OECD guidance No. 23.
The test item was carefully warmed-up at maximum 50 °C to melt the solid and possibly inhomogeneous test item. The molten test item was carefully mixed by repeated aspirating and dispensing of a pipette tip. After cooling to ambient temperature the test item was used for test media preparation.
For the two highest test loadings of 10 and 1 mg/L the required amounts of the test item were transferred into 2 L sterile glass flasks via sterilised Teflon weighing boats. The test item was carefully rinsed off using a pipette and the Teflon weighing boat was also introduced into the bottle. The flasks were brought to the volume of 1 or 2 L with sterile growth medium to obtain the required nominal concentrations. The test media were stirred for a few minutes and sonicated using an ultrasonic bath for 10 minutes to facilitate complete mixture and dispersion of the introduced test item. Immediately afterwards and after a check for homogeneous dispersion, the test media loaded with 1 mg/L was diluted with sterile growth media to obtain the other test loadings. This procedure was chosen since direct addition of test item amounts of 0.32, 0.1 and 0.01 mg/L was not possible, since these low amounts cannot be weighed out with a sufficient precision.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Origin: SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, D-37073 Göttingen, Catalog No 61.81 SAG
- Cultivation and pre-culture: The stock cultures are maintained fulfilling the criteria of the OECD guideline (culture medium recommended by Bringmann und Kühn (1980). Prior to testing a pre-culture was established in standard OECD growth medium to obtain exponentially-growing algae for the test. The culture duration of the pre-cultures was 3 days.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21.9 - 22.1 °C
pH:
test start: 7.87 - 8.03
test end: 7.79 - 7.89
Nominal and measured concentrations:
Nominal test item concentrations: 0.01, 0.10, 0.32, 1.0 and 10 mg/L
The measured C16-18 alcohol concentrations (GC-MS) were below the LOQ at test start and at test termination (LOQ of the GC-MS measuring solution = 2 μg/L).
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL conical glass flasks
- Type: covered with air-permeable siliconesponge caps
- Replicates of the control: 8
- Replicates of the test concentrations: 4
- Cells density: pre-culture (563 μL at a cell density of 1.78 x 10E6 cells/mL) was added to the test vessels to achieve a initial cell concentration of 10,000 cells/mL

GROWTH MEDIUM
- Standard medium used: Sterilised synthetic OECD medium according to OECD 201

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Light quality: OSRAM Standard “cool white” light adjusted to 4440-8880 lux
- Light intensity ranged from 98.4 to 100 μmol m-2 s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Electronic particle counter (CASY 1 Model TT, OMNI Life Science GmbH & Co. KG, Bremen, Germany).

RANGE FINDING STUDY
- Test concentrations: With three nominal loadings of 0.01, 0.1, 1.0 and 10 mg test item/L (without chemical analysis)
- The test media with loadings of 10 and 1 mg/L were filtrated prior to use for the algal test.
- The loadings of 0.1 and 0.01 were prepared by dilution of the 1 mg/L loading subsequent after its preparation and used without filtration.
- Results: After 72 hours exposure the inhibition of algal growth rate compared to controls was 8.3, 8.5, 32.9 and 28.4 % at nominal loadings of 0.01, 0.1, 1.0 and 10 mg test item/L. No clear concentration effect relationship was obtained.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Loading rate of a Water Accommodated Fraction (WAF)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Loading rate of a Water Accommodated Fraction (WAF)
Basis for effect:
growth rate
Details on results:
EL50 (72h) Yield: >10 mg/L (nominal)
NOEL (72h) Yield: >= 100 mg/L (nominal)
Results with reference substance (positive control):
ErL50 = 3.40 mg/L (3.32 - 3.48 = 95 % confidence limits), which is in good agreement with the results of an international ring test with ErL50 of 3.38 ± 1.30 mg/L (ISO 8692 Standardisation, 2010)

Treatment nominal

[mg test item/L]

% inhibition after 72 h

growth rate

yield

Control

0

0

0.01

-1.6

-7.6

0.10

-2.2

-10.9

0.32

-1.2

-5.7

1.00

-1.5

-7.4

10.0

-5.3

-29.0

- negative values indicate increase in the observed parameter

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented publication
Justification for type of information:
QSAR prediction
Principles of method if other than guideline:
Alcohol ethoxylte specific QSAR, with the log Kow as the predictor variable. The structure activity relationship was evaluated based on data of standardized ecotoxicity tests according to OECD guideline 201, with green alga Desmodesmus subspicatus.
GLP compliance:
no
Vehicle:
no
Details on test solutions:
Details on the tests which were used to evaluate the QSAR model:
-------------------------------------------------------------------------------
No solvent was used. Due to solubility limits some solutions were sonicated (ultrasound).
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Details on the test organisms which were used to evaluate the QSAR model:
-----------------------------------------------------------------------------------------------------------
- Source: Department of Plant Physiology, University of Goettingen, Germany
- Culturing and test media: Mineral salt media according to Directive 92/69/EWG, Annex C3 corresponding to OECD 201
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Details on test conditions:
Details on the tests which were used to evaluate the QSAR model:
-----------------------------------------------------------------------------------------------------------
- Light: 2000 Lux, full spectrum, constant illumination
- Initial inoculum: 1x10E4 cells/mL, in exponential growth phase
- Counting method: Calibrated Coulter counter, calibrated by manual cell counting before each counting routine
- Temperature: 23 +/- 2°C
- Shaker rotation: 100 rpm, horizontal shaker
- Frequency of counts: Every 24h (24, 48 and 72 h)
- Exposure concentrations were chosen on range finding tests
- Dilution series were constructed using factors between 2 to 3
- Results were statistically evaluated by exposure-response modeling (point estimate by regression) and threshold estimation (analysis of variance).
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.084 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.226 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
growth rate

Description of key information

EL50 (72h) > 10 mg/L for Pseudokirchneriella subcapitata (OECD 201)

ErC20 (72h) = 0.226 mg/L for Desmodesmus subspicatus (QSAR estimation, used for long-term assessment)

Key value for chemical safety assessment

Additional information

As there are no data available for alcohol ethoxylate C16, <2.5EO (CAS 9004-95-9) the acute toxicity to aquatic algae is assessed on the basis of data from similar alcohol ethoxylates. One reliable study is available on the toxicity of alcohol ethoxylate (C16-18, < 2.5 EO, CAS 68439-49-6) to algae (BASF, 2015). The study was performed according to OECD guideline 201 and GLP, with the unicellular freshwater green alga Pseudokirchneriella subcapitata as test species. Due to the very low water solubility of the substance, the test media were individually prepared by applying the Water Accommodated Fraction (WAF) approach according to the OECD guidance No. 23. The test design includes eight replicates of the control (test medium only) and four replicates of each of the five test concentration (0.01, 0.10, 0.32, 1.0 and 10.0 mg/L). The concentrations of the test item in the aqueous phase were determined by chemical analyses at the start and at the end of the test using GC-MS. The measured concentrations were below the LOQ in all test media at start and end of the test. Since the substance is a UVCB compound, the nominal loading was used for the test evaluation. There was no inhibition of algal growth up to the highest loading of 10 mg/L and the 72 hour ErL50 (based on specific growth rate) and EyL50 (based on algal yield) were estimated to be > 10 mg test item/L, in terms of the nominal loading. Additionally a category read-across is prepared to a study conducted with diethylenglycol – hexadecylether (C16E2, CAS 5274-61-3) (BASF, 1997). The study was conducted according to EU method C.3 and GLP with a limit test concentration of 100 mg/L. The unicellular green algae Scenedesmus subspicatus was used as test species. The nominal test concentration lays over the water solubility of the substance. The insoluble phase formed in the medium was filtered prior to test initiation. Verification of test concentrations was performed using gas chromatography. The test substance recovery by analytical methods was under the detection limit of 0.1 mg/L. No toxic effect of the substance was observed on algae. Therefore the EC50 (72h) for growth and yield are > 100 mg/L (nominal).

Since only a few data on the long-term aquatic toxicity for the mixtures within the category are available, the assessment on long-term aquatic toxicity is based for all category members on alcohol ethoxylate specific QSARs (Wind & Belanger, 2006; Boeije et al., 2006; Belanger et al, 2006). The QSARs considered all three trophic levels (algae, invertebrates and fish). The effects are described as EC20 values, since the QSARs are more robust for the EC20 values than for NOEC or EC10 values. For the described mixture of alcohol ethoxylate C16, <2.5EO (CAS 9004-95-9) the QSAR for long-term toxicity on green algae Desmodesmus subspicatus (Wind & Belanger, 2006) result in an EC20-value of 0.084 mg/L based on biomass and 0.226 mg/L based on growth rate, respectively. This result (ErC20 = 0.226 mg/L) is used as key value for the assessment on long-term effects.