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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:
2018-05-14 to 2018-05-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- CAS number: 39464-70-5
- Appearance: Clear, yellow liquid
- Batch No.: M-1025-157
- Purity: 100%
- Expiry date: 25 October 2018
- Storage conditions: Room temperature (15-25 °C, ≤ 70 % relative humidity (RH))
- Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100 mg/L
- Sampling method: Additional samplings for analysis at 24-hour intervals were used in order to better define the possible loss of the test substance during the exposure period. Sample from the Control was taken for analysis at the start of the test.
Vehicle:
yes
Remarks:
Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The Test Item is an UVCB and soluble in the test media, therefore a stock solution with the concentration of 100 mg/L (nominal) was prepared with Test Item and test medium (OECD medium) by direct addition, with mixing and using approximately ultra-sonication as needed. As only a Limit test was carried out, further dilution of this stock solution was not performed. Test solution was distributed into test vessels prior to introduction of algae.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of Citoxlab Hungary Ltd.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Remarks on exposure duration:
The cell concentration were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
Test temperature:
between 21 °C and 24 °C with a maximum deviation of ± 2 °C
pH:
6.94 – 8.96
Nominal and measured concentrations:
100 mg/L (nominal and measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Aeration: Flasks covered with air-permeable stoppers
- Initial cells density: 10E+4 algal cells per mL test medium
- No. of vessels per concentration (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted algal growth medium (OECD medium, according to OECD 201) will be used as the dilution medium in the experiment.

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to not vary by more than 1.5 units
- Photoperiod: Continuous lighting by fluorescent lamps (spectral range of 400-700 nm)
- Light intensity and quality: 4440-8880 lux (mean value +/- 15% variation) at the liquid surface
- Salinity (for marine algae): n.a.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: microscopic method with a counting chamber

TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations during range-finding test: 0.1, 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: Yes, because significantly toxic response was not observed during the preliminary concentration Range-Finding Test, only one test concentration at 100 mg/L (nominal) and one Control group were tested in a Limit Test.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (tested twice a year)
Key result
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: As the measured concentrations di not deviate more than ± 20% of the nominal, therefore the biological results are based on the nominal concentrations
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: As the measured concentrations di not deviate more than ± 20% of the nominal, therefore the biological results are based on the nominal concentrations
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: As the measured concentrations di not deviate more than ± 20% of the nominal, therefore the biological results are based on the nominal concentrations
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: There were no morphological deviations of the algal cell during the study in any of the groups. All cells were considered normal.
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- ErC50: 0.88 mg/L, (95 % confidence limits: 0.81 – 0.96 mg/L)
- Other: For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions. The date of the last study (Study Code: 17/367-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704):
04–07 December 2017.
The 72h ErC 50: 0.88 mg/L, (95 % confidence limits: 0.81 – 0.96 mg/L)
The 72h EbC 50: 0.63 mg/L, (95 % confidence limits: 0.58 – 0.69 mg/L)
The 72h EyC 50: 0.53 mg/L, (95 % confidence limits: 0.49 – 0.58 mg/L)

These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).
Reported statistics and error estimates:
The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period.
The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).


The ErC50 and EyC50 values of the Test Item were determined from the raw data.
For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by 2 Sample t-Test (alpha = 0.05) by TOXSTAT software.

Average Specific Growth Rates

The results of the statistical evaluation (based on 2 Sample t-Test; alpha= 0.05) showed that the 0-72 h average specific growth rate was not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) was determined as 100 mg/L (nominal).

 

Table 1: Growth Rates (µ) and Percentage Inhibition of µ during the Test Period

Test group

Growth rate and % inhibition of µ

0–24 h

0–48 h

0–72 h

µ

%

µ

%

µ

%

Control

0.0573

0.0

0.0595

0.0

0.0589

0.0

100 mg/L (nominal)

0.0553

3.5

0.0596

-0.1

0.0586

0.5

AREAS UNDER THE GROWTH CURVES

The results of the statistical evaluation (based on 2 Sample t-Test; alpha= 0.05) showed that the 0-72 h areas were not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) was determined as 100 mg/L (nominal).

 

Table 2: Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Test group

Areas under the Growth Curves (A) and % inhibition of A

0–24 h

0–48 h

0–72 h

A

%

A

%

A

%

Control

36.0

0.0

270.0

0.0

1290.0

0.0

100 mg/L (nominal)

34.0

5.6

266.0

1.5

1268.0

1.7

Yield

The results of the statistical evaluation (based on 2 Sample t-Test; alpha= 0.05) showed that the 0-72 h yield was not statistically significantly different from the untreated control value; accordingly, the No Observed Effect Concentration (NOEC) was determined as 100 mg/L (nominal).

 

Table 3: Yield (Y) and Percentage Inhibition of Y during the Test Period

Test group

Yield (Y) and % inhibition of Y (0-72 h)

Y

%

Control

68.5

0.0

100 mg/L (nominal)

67.0

2.2

Validity criteria fulfilled:
yes
Conclusions:
In a Growth Inhibition Test conducted according to the OECD guideline 201, a 72-hr ErC50 of Ethoxylated phenol phosphate to green algae (Pseudokirchneriella subcapitata) was determined to be above 100 mg/L as no significant effect was observed for cultures of P. subcapitata exposed to 100 mg/L of the test item.
Executive summary:

In a 72-hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata were exposed to Ethoxylated phenol phosphate at nominal concentrations of 100 mg/L under static conditions in accordance with the OECD Guideline 201 (Alga, Growth Inhibition Test), EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II), and EU Method C.3 (Algal Inhibition test).  The EC50 value based on cell density was > 100 mg/L. All nominal Test Item concentration could be quantified during the study. There was no Test Item detected in the Control samples. The measured concentrations did not deviate more than ± 20% of the nominal, therefore the biological results are based on the nominal concentrations.

There were no compound related phytotoxic effects.

 This toxicity study is classified as acceptable and satisfies the guideline requirements for the Alga growth inhibition toxicity study.

 

Results Synopsis

 

Test Organism: Pseudokirchneriella subcapitata

Test Type (Flowthrough, Static, Static Renewal): Static

 

The 72h EbC50value: >100 mg/L (nominal)

The 72h ErC50value: >100 mg/L (nominal)

The 72h EyC50value: >100 mg/L (nominal)

The 72h NOEC:  100 mg/L (nominal)

The 72hLOEC: >100 mg/L (nominal)

Description of key information

In a Growth Inhibition Test conducted according to the OECD guideline 201 the freshwater algae Pseudokirchneriella subcapitata were exposed to 100 mg/L Ethoxylated phenol phosphate. During this test no effects were observed on the yield or growth rate. A 72-hr ErC50 was therefore reported to be above 100 mg/L. The measured concentrations did not deviate more than ± 20% of the nominal, therefore the biological results are based on the nominal concentrations.

The 72h EbC50value: >100 mg/L (nominal)

The 72h ErC50value: >100 mg/L (nominal)

The 72h EyC50value: >100 mg/L (nominal)

The 72h NOEC: 100 mg/L (nominal)

The72h LOEC: >100 mg/L (nominal)

Key value for chemical safety assessment

Additional information