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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:
13 - 16 Mar 2017
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)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Staatliches Gewerbeaufsichtsamt Hildesheim, Germany (03 Jan 2017).
Analytical monitoring:
yes
Remarks:
GC-MS
Details on sampling:
- Concentrations: Control, 0.255, 0.806, 2.55, 8.058, and 25.5 mg/L (nominal concentrations) after 0 h and 72 h of exposure.
- Sampling method: Separate replicates for each test item concentration and control were prepared with algae for the test item analysis at the beginning of the exposure. The sampels for the test item analysis after 72 h were prepared with algae and incubated under test conditions.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until sample preparation and at room temperature until the start of the analysis (on an autosampler), if necessary. Prepared extracts were stored at 6 ± 2 °C for potential reanalyses.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A saturated solution with a nominal concentration of 25.5 mg/L of the test item was freshly prepared with dilution water 72 ± 1 h prior to the start of the exposure phase in a tightly closed glass flask (screw cap with a septum), which was filled up with dilution water without leaving headspace (total volume: 2275 mL) and 39.4 g glass pearls. The test item was pipetted onto a cover slip and inserted into the brown glass flask. This dispersion was shaken overhead for at least 72 ± 1 h with 20 rpm at room temperature. The glass pearls were used to enhance mixing. After a separation phase of 1 h the dispersion was removed by siphoning from the bottom . The solution was checked after stirring via laser beam (Tyndall effect) for undissolved test item, which was positive. The saturated solution and four dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 10^(1/2), i.e.: 1.00, 3.16, 10.0, 31.6, and 100% of the saturated solution.
- Controls: Six replicates (without test item) were incubated under the same conditions as the test concentrations.
- Evidence of undissolved material: The solution was checked after stirring via laser beam (Tyndall effect) for undissolved test item, which was positive.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: unicellular freshwater green microalgae
- Strain: SAG 61.81
- Age of inoculum (at test initiation): A three day old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-agar. Light intensity is 2567 - 5130 lux for 24 h/d.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23.0 °C (mean)
pH:
7.50 - 7.53 (0 h)
7.56 - 8.73 (72 h)
Nominal and measured concentrations:
1.00, 3.16, 10.0, 31.6, and 100% of saturated solution
0.128, 0.457, 1.43, 5.16, and 17.2 mg/L (geometric mean measured concentration)
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile headspace flasks
- Type: Closed with aluminium tops with PTFE seals.
- Material, size, headspace, fill volume: Size: 59 mL; Headspace: none; Fill volume: 59 mL
- Initial cells density: 6839 cells/mL
- Control end cells density: 500377 cells/mL
- No. of vessels per concentration (replicates): 3. Separate replicates for each measuring time were prepared.
- No. of vessels per control (replicates): 6. Separate replicates for each measuring time were prepared.

GROWTH MEDIUM
- Standard medium used: Yes, nutrient medium Z, according to Lüttge et al. (1994)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Threefold concentrated AAP medium according to the guidelines.
- The pH of the dilution water has to be 7.5 ± 0.1 and may be adjusted prior to testing with addition of 1 M NaOH or HCl.
- Culture medium different from test medium: Yes, culture medium was different from test medium: A three day old preculture was prepared in dilution water (3-fold AAP medium). Algae were cultivated in nutrient Z medium.
- Intervals of water quality measurement: At the start of exposure, pH-values were measured in one additional replicate of each test item concentration and the control. At the end of exposure, the pH-values were measured in pooled samples of each test item concentration and the control. Room temperature was measured continuously. Light intensity was measured prior to the start of exposure.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the dilution water has to be 7.5 ± 0.1 and may be adjusted prior to testing with addition of 1 M NaOH or HCl.
- Photoperiod: 24 h/d
- Light intensity and quality: Mean 5812 lux, within ± 15% over incubation area
- Other: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm. The flasks were positioned randomly and repositioned daily.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Fluorometer: Microplate Reader Chameleon V (Hidex) with Software Micro Win 4.41 (Mikrotec Laborsysteme GmbH)
- Chlorophyll measurement: Daily measurements (excitation wavelength: 436 nm, emission: 685 nm)
- Microscopic evaluation: At the start and the end of the incubation period, the cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10^(1/2) = approximately 3.162
- Range finding study: Yes, preliminary range finding test (non-GLP, closed bottles without headspace).
- Test concentrations: 0.1, 1, 10, and 100% of saturated solution.
- Results used to determine the conditions for the definitive study: Yes: 0.1% of saturated solution: 1% growth rate inhibition; 100% saturated solution: 99% growth rate inhibition.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.01 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 0.858 - 1.18 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
6.49 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 6.14 - 6.89 mg/L
Details on results:
- Exponential growth in the control: Yes: 73-fold (1.43 day^-1, specific growth rate)
- Observation of abnormalities: No morphological abnormalities were observed.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The saturated solution was checkked after stirring via laser beam (Tyndall effect), which was positive.
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes, the reference item toxicity is in the valid range of the test facility SOPs (ErC50 (72 h): 0.782 ± 0.534 mg/L).
- ErC50 (72 h): 0.435 mg/L, 95% confidence interval: 0.414 - 0.458 mg/L (with headspace); 0.811 mg/L, 95% confidence interval: 0.763 - 0.883 mg/L (without headspace)
- Other: The toxicity of potassium dichromate was determined from 11 - 14 Oct 2016 with and without headspace.
Reported statistics and error estimates:
Calculations were carried out using Excel (Microsoft Corp.), SigmaPlot (SPSS Inc.), ToxRat (ToxRat Solutions GmbH), GraphPad Prism (GraphPad Software Inc.).

The EC10, EC20 and EC50 values with confidence intervals of growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression.
The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield. The following statistical tests were conducted:
- Monotonicity was tested by trend analysis by contrasts (significance level 0.05)
- Shapiro-Wilk´s test on normal distribution was done with a significance level of 0.01.
- Levene´s test on variance homogeneity was done with a significance level of 0.01.
- William´s test on significant differences was done for yield with a significance level of 0.05.
- Step-down Jonckheere-Terpstra test procedure was done for growth rate with a significance level to 0.05.

ANALYTICAL RESULTS

 

The measured concentrations of the test item at the start of exposure (0 h) were in the range of 63 – 72% of the nominal test item concentrations and at the end of the exposure (72 h) in the range of 49 – 105% of the measured initial values. The geometric mean measured concentrations are: 0.128, 0.457, 1.43, 5.16, and 17.2 mg/L

 

Table 1. Measured concentrations of the test item during the definitive test.

 

 

 

Nominal test item concentration

0 h

Fresh medium

72 h

Old medium

 

 

Measured conc. [mg/L]

 

Measured conc. [mg/L]

 

%

Geometric mean measured test item concentration [mg/L]

25.5

17.7

16.6

94

17.2

8.058

5.04

5.28

105

5.16

2.55

1.67

1.22

73

1.43

0.806

0.5491)

0.381

69

0.457

0.255

0.183

0.08952)

49

0.128

Control

< LOQ

< LOQ

< LOQ

Meas.conc.       = measured concentration of the test item (dilution factor taken into account)

%                         = percentage of the initially measured concentration of the test item

LOQ                     = Limit of quantification (0.01 mg test item/L)

1)                        = Reanalyzed 2 d later, mean value of two replicates

2)                         = Reanalyzed due to technical reasons 7 d later of the extracted and derivatized samples: the samples were considered stable, therefore occurred no conflict with the expiry date of the test item. (A potential degradation of the derivatized product is alleviated since the derivatization of calibration levels and the test level concentration occurred on the same day. If an aging of the samples occurs, calibration levels and test concentrations will show the same aging effect.)                    

 

BIOLOGICAL RESULTS

Table 2. Evaluation after 72 h. Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Geometric mean measured test item concentation [mg/L]

Replicate n°

Growth rate [d^-1]

Inhibition of growth rate [%]

 

17.2

1

-0.288

100

2

< LOQ

100

3

< LOQ

100

mean

(+)            n.a.

100

 

5.16

1

0.846

41

2

0.840

41

3

0.820

43

mean

(+)          0.835

42

 

1.43

1

1.20

16

2

1.28

11

3

(+)           1.23

14

mean

1.23

14

 

0.457

1

1.38

4

2

1.36

5

3

1.35

5

mean

(+)           1.36

5

 

0.128

1

1.40

2

2

1.43

0

3

1.39

3

mean

(-)            1.41

2

 

 

 

Control

1

1.44

 

2

1.43

 

3

1.48

 

4

1.43

 

5

1.39

 

6

1.42

 

Mean

1.43

 

< LOQ                 = below limit of quantification

n.a.                     = data not applicable

Validity criteria fulfilled:
yes
Conclusions:
An ErC10 (72 h) of 1.01 mg/L and an ErC50 (72 h) of 6.49 mg/L were obtained for the toxicity to aquatic algae.
Executive summary:

The toxicity to aquatic algae was assessed in static test according to OECD guideline 201. P. subcapitata was exposed for 72 h to four dilution levels with a spacing factor 3.16 of a saturated solution prepared with a nominal loading rate of 25.5 mg test item/L. The test item concentrations wree analytically verified at the beginning and end of the test.

The measured initial concentrations ranged from 0.183 to 17.7 mg/L. The measured concentrations at the end of the test were 49 - 105% of the initially measured concentrations. The test resulted in an ErC10 (72 h) of 1.01 mg/L and an ErC50 (72 h) of 6.49 mg/L.

Description of key information

ErC10 (72 h) = 1.01 mg/L (geometric mean measured, P. subcapitata, OECD 201)

ErC50 (72 h) = 6.49 mg/L (geometric mean measured, P. subcapitata, OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:
6.49 mg/L
EC10 or NOEC for freshwater algae:
1.01 mg/L

Additional information

There is one study available, in which the toxicity of the substance to aquatic algae was investigated according to OECD guideline 201 and GLP.

In a static test, P. subcapitata at an initial cell density of 6839 cells/mL was exposed to 1.0, 3.16, 10.0, 31.6, and 100% of a saturated solution with a nominal loading of 25.5 mg/L test item for 72 h. The test item concentrations were analytically verified by GC-MS at the beginning (0 h) and at the end (72 h) of the test.

The measured initial concentrations (at 0 h) were in the range of 63 to 72% of the nominal test item concentrations and at the end of the exposure (72 h) in the range of 49 to 105% of the measured initial values. The geometric mean measured concentrations are 0.128, 0.457, 1.43, 5.16, and 17.2 mg/L.

After 72 h, the test item was found to inhibit the growth of the freshwater green algae and the following effect concentrations were obtained: ErC10 (72 h) = 1.01 mg/L and ErC50 (72 h) = 6.49 mg/L (geometric mean measured concentration).

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