Registration Dossier

Diss Factsheets

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:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Storage conditions: Samples were stored deep frozen and protected from light unti they were analysed
Analytical monitoring:
yes
Remarks:
Gas Chromatographic (GC)
Details on sampling:
- The stability ot test concentration during exposure was examined by chemical analysis at 0 and 72 hours.
- Samples of stock solution and test solutions were taken to determine the actual test item concentrations in comparison to the nominally applied concentrations.
- Control and test solutions were sampled in duplicate. The duplicate samples were kept separately as a reserve. The volume of each sample was recorded.
- Samples of a defined volume (10 mL) were taken from the designated test solution. After sampling and before shipment, the samples were stabilised by adding acetonitrile directly after sampling (10 mL of sample were mixed with 10 mL of acetonitrile).
- After sampling and before shipment, all samples were stored in glass bottles in the dark at a temperature of -18°C if not advised otherwise. A record was kept for each sample.
Vehicle:
no
Details on test solutions:
Preparation of Stock, Application and Test Solutions:
- A stock solution at a nominal concentration of 100 mg test item/L was prepared by directly adding 100.7 mg test item to 1000 mL test medium, stirred using a
magnetic stirrer at 700 rpm for 24 min at ambient temperature in the dark and left to settle for 54 minutes; visual examination showed a clear stock solution
without undissolved/particulate matter; the pH of the stock solution was adjusted with 1 M Ha to 8.3
- The stock solution was prepared once to prepare the test solutions
- The volume of the stock solution was large enough to prepare the test solutions and all analytical samples and solutions used for conditioning
- The vessels containing the test solutions were stirred for ca. 5 minutes on a magnetic stirrer at room temperature and placed into a temperature controlled room for temperature adaptation.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Species: Pseudokirchneriella subcapitata (SAG 61.81), currently valid species name: Raphidocelis subcapitata
- Supplier: Sammlung von Algenkulturen, Albrecht-von-Haller-Institut, Universität Göttingen, Germany
- Date of receipt: February 24, 2016
- Charge No. (ECT): A/240216/A
- pH-value of the algal medium: 8.3
- Culture medium: Mod. OECD medium (OECD 201, EN ISO 8692)
- Volume of liquid stock culture per pre-culture vessel: 100 ± 5 mL
- Pre-culture vessels: 300 mL Erlenmeyer flasks
- Number of replicates: 2
- Light: Permanent illumination (24/0 h light/dark); Econlux LED Sunstrip "daylight"
- Light intensity: 60-120 pE m-2s-1; (measured: 71.0-71.4 pE m-2s-1)
- Shaker: 100±5 oscillations/min
- Temperature in the test room: 21-24 °C
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
No data
Test temperature:
21.8-22.0 °C
pH:
7.6 - 7.8
Dissolved oxygen:
n.a.
Salinity:
n.a.
Conductivity:
no data
Nominal and measured concentrations:
Test concentrations (nominal): 6.25, 12.5, 25.0, 50.0 und 100 mg test item/L
The following geometric mean concentrations were determined: 9.30, 14.7, 24.3, 42.7, and 71.6 mg/L
Details on test conditions:
EXPOSURE CONDITIONS:
- Test vessels: Glass Erlenmeyer flasks (300 mL) covered by air-permeable lids
- Volume of test solution per test vessel: 100±5 mL
- Age of the pre-culture: 4 days
- Number of cells per mL in the pre-culture before inoculating the test solutions: 95.5x10E+04
- Number of cells per mL test solution at the beginning of the test: 0.3x10E+04
- Number of replicates per test item concentration: 3
- Number of replicates in the control: 6
- Number of replicates for stability check (highest concentration level without algae): 1 (without algae)
- Test concentration: 5 (6.25, 12.5, 25.0, 50.0 und 100 mg test item/L)
- Test medium: OECD medium (modified)
- pH of test medium: 8.0
- pH in test solutions: 7.6-7.8
- Light regime: 24 h light/0 h dark
- Type of light: Econlux LED Sunstrip "daylight"
- Light intensity: 78.5-87.4 pE m 2s-1; mean 83.7 pE m 2s-1
- Temperature: 21.8-22.0, mean 21.9 °C
- Shaker: 100±5 oscillations/min; the test vessels were placed randomly on a horizontal shaker
- Counting of algae: Daily

WATER QUALITY MEASUREMENTS:
- After temperature adaptation of the test solutions, the pH was measured and recorded for each test concentration and the control
- Temperature was recorded once per hour throughout the test in a separate test vessel placed on the shaker containing approximately 100±5 mL deionised water
- At the end of the test, the pH was measured and recorded in the pooled replicates per test concentration and control
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 95.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
95.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
42.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
71.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The results are expressed in terms of nominal concentrations and geometric mean measured values. Effective concentrations ranged from 0.41 to 0.46 % of nominal values at 0 hours, corresponds 0.15 % of nominal values at 24 hours, from 0.12 % to 0.15 % of nominal values at 48 hours and from 0.07 % to 0.19 % of nominal values at 72 hours.
Results with reference substance (positive control):
Standard: Growth rate ErC50 (0-72 h): 0.952 mg/L (0.9160-0.990 mg/L; 95%-CL).

The ErC50 value for the toxic reference item, potassium dichromate, was determined as 0.952 mg/L. This value is within the range of the international ring test (ISO (2004)). Therefore the results of this reference test are acceptable and the test conditions are reliable.
Reported statistics and error estimates:
DATA EVALUATION:
- Shapiro-VVilk's Test on Normal Distribution;
- Levene's Test on Variance Homogeneity; Williams
- Multiple Sequential t-test and Welsh-t test After
- Bonferroni-Holm for homogeneous variances
- Probit and Logit analysis
Validity criteria fulfilled:
yes
Remarks:
Factor of the biomass parameter (305.2), measured in the control between 0 and 72 h, was at least 16; the mean coefficient of variation (5.9%) was not higher than 35%; the coefficient of variation (0.6%) of the mean specific growth rate did not exceed 7%
Conclusions:
In this toxicity test to Pseudokirchneriella subcapitata according to OECD 201 an EC 50 value of higher than 95 mg/L and a EC10 value of 95 mg/L mg/L were obtained after 72 hours of exposure. The results are expressed in terms of geometric mean measured values. At time 0 hours the measured concentrations ranged between 76.9-170.6% of the nominal concentrations and 66.7-129.8% after 72 hours.
Executive summary:

The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 'Alga, Growth Inhibition Test' (2006). The purpose of this method was to determine those concentrations which caused a 10 % and a 50 % adverse effect (EC 10, EC 50) or, if conducted as a limit test, to determine the adverse effects at a maximum test concentration of 100 mg/L or at the limit of water solubility. This study was conducted under static conditions; the algae were exposed to a test solution which was not be renewed during the test. Based on the results of a preliminary non-GLP range finding test, the following concentrations of 6.25, 12.5, 25.0, 50.0 und 100 mg test item/L in a geometrical series were tested in the main test. The data obtained were analysed in order to estimate the concentration that caused a x% growth inhibition, i.e. EC x (e.g. EC50, EC20 or EC10). As additional endpoints the No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) were calculated. Growth was determined daily based on determination of the cell number per volume test solution (cell concentration). To verify the nominally applied concentration, samples were taken from the test solution and analytically measured. The samples taken from the test vessels were transferred to the analytical test site and analysed under the responsibility of the Principal Investigator for analysis. During the test a temperature range of 21 -24 °C was maintained in the test vessels. The pH was measured at the beginning of the test and after 72 hours of exposure. In this toxicity test to Pseudokirchneriella subcapitata according to OECD 201 an EC 50 value of higher than 95 mg/L and a NOEC value of 42.7 mg/L were obtained after 72 hours of exposure. The results are expressed in terms of geometric mean measured values. At time 0 hours the measured concentrations ranged between 76.9-170.6% of the nominal concentrations and 66.7-129.8% after 72 hours. All validity criteria were fulfilled as required. This toxicity study is classified as acceptable and satisfies the guideline requirements for the Algae study.

Description of key information

In this toxicity test to Pseudokirchneriella subcapitata according to OECD 201 an EC 50 value of higher than approx. 95 mg/L and a EC10 value of 95.1 mg/L were obtained after 72 hours of exposure. The results are expressed in terms of geometric mean measured values. At time 0 hours the measured concentrations range between 76.9-170.6% of the nominal concentrations and 66.7-129.8% after 72 hours

Key value for chemical safety assessment

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

Additional information

"Should read: 72 h-EC 50 (growth rate): >95.1 mg/L (The results are expressed in terms of geometric mean measured values)"

Categories Display