Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 24, 2018 - September 26, 2018
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:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 17083101
- Expiration date of the lot/batch: August 30, 2019
- Purity: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, at 20 ± 5 °C. Keep container tightly closed in a dry and well-ventilated place.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 6.25, 12.5, 25, 50 and 100 mg/L.
- Sampling method: Duplicate samples were taken from the test media of all test concentrations and the control at the start of the test (without algae) and at the end of the test (containing algae). For sampling at the end of the test, the test medium of the treatment replicates was pooled. The concentrations of the test item were determined in one of the duplicate samples from the nominal test concentrations of 50 and 100 mg/L from both sampling times (0 and 72 hours). The samples from the nominal test concentrations of 6.25 to 25 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test and thus not relevant for the interpretation of the biological results. From the control samples, one of the duplicate samples was analyzed per sampling time.
- Sample storage conditions before analysis: Immediately after sampling, all samples were frozen (at -20 ± 5 °C). In pre-experiments for investigation of the storage stability of the samples (IES non-GLP Study 20180007), the test item was found to be stable under these storage conditions
Vehicle:
no
Details on test solutions:
- Method: The test medium of the highest nominal concentration of 100 mg/L was prepared by dissolving 100.0 mg of the test item completely into 1000 mL of test water by using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature. After the dissolving procedure the highest test concentration of 100 mg/L was a clear solution. The highest test concentration of 100 mg/L was further diluted with test water to prepare the test media with the lower test concentrations. The test media were prepared just before the start of the test.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: No. 61.81 SAG
- Source (laboratory, culture collection): cultivated at IES Laboratories under standardized conditions according to the test guidelines
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.

ACCLIMATION
- Acclimation period: not relevant (except pre-culture 3 days before start of the test)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
0.15 mmol/L (= 15 mg/L as CaCO3)
Test temperature:
23 °C
pH:
test start: 7.5
test end: 8.9
Nominal and measured concentrations:
Range-finder:
nominal test concentrations: 1.0, 10 and 100 mg/L
measured test concentration t = 0 h: 0.95, 10.8, 87.4 mg/L
measured test concentration t = 72 h: 0.84, 9.69, 87.6 mg/L

Final test:
nominal test concentrations: control, 6.25, 12.5, 25, 50 and 100 mg/L.
measured test concentration t = 0 h: n.d., 47,3, 95,6 mg/L
measured test concentration t = 72 h: n.d.,47.7, 96.1 mg/L
* The samples from the nominal test concentrations of 6.25 to 25 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test and thus not relevant for the interpretation of the biological results. From the control samples, one of the duplicate samples was analyzed per sampling time.

* Measured concentrations remained stable until the end of the exposure (98-102% of the initial concentrations).
Details on test conditions:
TEST SYSTEM
- Test vessel: flasks
- Type (delete if not applicable): Erlenmeyer
- Material, size, headspace, fill volume: Each test flask was covered with a glass dish. The volume in each test flask was 30 mL per replicate.
- Initial cells density: 5,000 cells/mL
- Control end cells density: not reported
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Reconstituted test water (AAP Medium) prepared according to the test guidelines. An inoculum culture was set up four days before the start of the exposure. The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water (AAP Medium) prepared according to the test guidelines
- Culture medium different from test medium: No.
- Intervals of measurements: Light intensity was measured at the start of the test. The temperature in the incubator was monitored and recorded continuously. The pH was measured in each treatment at the start and end of the test. The appearance of the test media was visually controlled and noted daily during the exposure period.

OTHER TEST CONDITIONS
- Sterile test conditions: no information
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: LED light installed above the test flasks. Light intensity at the level of the test solutions was approximately 71 µE s-1 m-2 (range: 67 to 75 µE s-1 m-2, measured at nine places in the experimental area). The light intensity over the incubation area was within a ±15 %-deviation from the average light intensity as recommended by the guideline.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations:A small volume (100 µL per sampling) of the algal suspension was withdrawn daily from each test flask for the measurement of the biomass, and was not replaced. The algal biomass in the samples was determined by fluorescence measurement (SpectraMax I3x, Molecular Devices Ltd, Wokingham Berkshire/UK). The measurements were performed at least in duplicate at an excitation of 440 nm and emission of 680 nm. At the end of the test, a sample was taken from the control and from the highest nominal test concentration tested of 100 mg/L to determine a potential influence of the test item on the algal cells. The shape and size of the algal cells were visually inspected.
- Effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations: control, 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.
- EC10 based on biomass an growth: > 100 mg/L
- EC20 based on biomass an growth: > 100 mg/L
- LOEC based on biomass an growth: > 100 mg/L
*95% confidence intervals could not be determined
*Inhibition of growth rate was 1.4 % at 100 mg/L
*Inhibition of yield was 7.3 % at 100 mg/L
Results with reference substance (positive control):
- Results with reference substance valid?
- 72-h EC50 for growth rate was 1.3 mg/L (April 2018, IES Study Number 20180052) and showed that the sensitivity of the test system was within the range recommended by the guideline (72-hour EC50 for the growth rate 0.92 - 1.5 mg/L).
Reported statistics and error estimates:
The 72 -hour EC10, EC20 and EC50 values for the inhibition of growth rate and yield could not be determined because of the low inhibitory effect of the test item on the algal growth at the tested concentrations. Therefore ECx values were directly determined from the Raw Data.
For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams’ t test. Statistical analysis was performed using ToxRat Professional.
Validity criteria fulfilled:
yes
Conclusions:
The study of Eckenstein (2018), investigating the acute toxicity of the test item to algae according to OECD guideline 201, was considered as the key study for endpoint coverage.
In conclusion, the test item had no toxic effects on Pseudokirchneriella subcapitata during the 72 hour test period up to and including the nominal concentration of 100 mg/L.
Executive summary:

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72‑hour static test according to the OECD Guideline 201 (2006, corrected 2011) and theCommission Regulation (EU) No. 2016/266, C3.

 

The nominal test item concentrations tested were 6.25, 12.5, 25, 50 and 100 mg/L. A control (test water without test item) was tested in parallel.

The measured concentrations of the test item in the test media of the test concentrations of 50 and 100 mg/L were 95 and 96 % of the nominal values, respectively, at the start and the end of the test. The samples from the nominal test concentrations of 6.25 to 25 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test and thus not relevant for the interpretation of the biological results.

Thus, the correct dosing of the test item was confirmed. The test item was stable in the test media over the test period of 72 hours. The biological results were related to the nominal concentrations of the test item.

The biological results of the study were as follows:

 Endpoint after 72 hours Growth rate (mg/L)  Yield (mg/L) 

EC10

95% Confidence Interval

>100*

n.d. 

>100**

n.d. 

EC20 

95% Confidence Interval

>100*

n.d. 

>100**

n.d. 

EC50 

95% Confidence Interval

>100*

n.d. 

>100**

n.d. 

 NOEC

≥100

≥100

 LOEC

>100

>100

n.d.: Could not be determined

*:  Inhibition of growth rate was 1.4 % at 100 mg/L

**:Inhibition of yield was 7.3 % at 100 mg/L

In conclusion, the test item had no toxic effects on Pseudokirchneriella subcapitata during the 72‑hour test period up to and including the nominal concentration of 100 mg/L.

Description of key information

The study investigating the acute toxicity of the test item to algae according to OECD guideline 201.

The test item had no toxic effects on Pseudokirchneriella subcapitata during the 72 hour test period up to and including the nominal concentration of 100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

The test item is not toxic on algae.