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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23-07-2016 to 29-07-2016 and 11-08-2016 to 22-09-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
At start of the test duplicate samples from all test concentrations and the control were taken
After 72hrs test duplicate samples from all test concentrations and the control were taken
All samples were stored in a freezer (< -20°C) at test facility.
By default for all concentration one replicate was sent to the analytical determination lab while the other was kept at
the biological test site. As for two probes, issues came up with the send replicate, the other replicate were send as well (This referred to probe with
highest concentration and the probe with the second highest concentration)
Vehicle:
yes
Details on test solutions:
As test water reconstituted water was used
Macro nutrients (mg/L):
NaHCO3 50 mg/L
CaCI2*2H20 18 mg/L
NH4Cl 15 mg/L
MgSO4*7H20 15 mg/L
MgCl2*6H20 12.0 mg/L
KH2PO4 1.6 mg/L

Trace elements (µg/L):
Na2EDTA*2H20 100
FeCl3 *6H20 64
MnCl2*4H20 415
H3BO3 185
Na2MoO4 *2H2O 7
ZnCl2 3
CoCl2*6H20 1.5
CuCl2*2H20 0.01

Calculated water hardness 0.24 mmol/L as caCO3
pH value was adjusted with 1M Hcl to pH 8.1

test concentrations: load ing of 100 mg/L of test item and dilutions with 1:2.5, 1:6.25, 1:15.6, 1:39.1 with nominal concentrations of 100, 40,16, 6.4 and 2.56 mg test item/L

Dosage of test item: before test start test concentration with highest loading was prepared by mixing 100.3mg test item with 1003ml test water. This solution was ultrasonicated for 10 minutes and stirred for 24 hours at room temperature in the dark. After 24 hours the undissolved part was seperated by filtration (0.45µm cellulose acetate filter). For the filtrate pH value was readjusted from pH 10.1 to pH 8.1. The remaining filtrate was used as the test item with the highest concentration and the other test items (with lower concentrations) were generated by dilution thereof.

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: Pseudokirchnerella subcapitata (formerly known as Selenastrum capricornutum, recently renamed as Raphidocelis subcapitata, strain no 61.81 SAG
- Origin (laboratory, culture collection): laboratory (Sammlung von Algenkulturen, Albrecht-von-Haller-Institut fürPflanzenwisssenschaften, Universität Göttingen)
breeding conditions: test laboratory under standardized conditions according to OECD guideline
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
from test water calculated 24mg/L as CaCO3
Test temperature:
21 to 23°C
pH:
8.1 at test start and 7.8 to 9.1 at test end (10.0 prior to adjustment of highest concentration)
Nominal and measured concentrations:
nominal conc. test item 100 | 40 | 16| 6.4 | 2.56 | 0 mg/L
measured conc. barium 0 h 5.94 | 2.49 | 1.10 | 0.44 | 0.18 | < 0.10 mg/L
measured conc. barium 72 h 6.66 | 2.60 | 1.07 | 0.42 | 0.18 | < 0.10 mg/L

measured conc. strontium 0 h 24.64 | 9.35 | 3.79 | 1.57 | 0.60 | < 0.10 mg/L
measured conc. strontium 72 h 24.22 | 9.54 | 3.81 | 1.58 | 0.60| < 0.10 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks of 50 ml volume with 50ml of test medium, 3 replicates per test concentration and 6 per control
- Type (delete if not applicable): flasks covered with glass dishes
- Initial cells density: 5000 cells/mL (taken from exponentially growing pre-culture, which was setup 4 days before test start under same conditions as test)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

Light regime and intensity: continuous illumination. Light intensity was measured once during test at 6 different positions at the test surface with mean lightintensity of 5292 lux ( range 5000-5720 lux)
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
60.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CI 95% 59.3-62.7
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
16.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CI 95% 15.3-17.2
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
6.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield and growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield and growth rate
Results with reference substance (positive control):
The sensitivity of the test system has to be demonstrated periodically using potassium dichromate.
Under the given experimental conditions, the test item potassium dichromate showed 72h EC50 values which are within the recommended range (Yield EC50 0.721 mg test item/L (CI 95% 0.588 – 0.847), biomass EC50= 0.794 mg test item/L (CI 95% 0.668-0.910) and Growth rate EC50=1.50 mg test item/L CI 95%( 1.43 –1.58 mg/L)). EC20 values are: Yield EC20 0.531 mg test item/L (CI 95% 0.367 – 0.638 mg/L), biomass EC20= 0.552 mg test item/L (CI 95% 0.381-0.658) and Growth rate EC20=0.731mg test item/L CI 95% (0.673 –0.785 mg/L)), EC10 values are : Yield EC10 0.452 mg test item/L (CI 95% 0.280 – 0.562), biomass EC10= 0.456 mg test item/L (CI 95% 0.278-0.569) and Growth rate EC10=0.502 mg test item/L CI 95% (0.447 –0.554 mg/L)),

Table 1: Yield and percentage inhibition of y during test period

 loading rate

[mg test item/L]

yield [10000/mL]@24 hrs

  

% inhibition

   

yield [10000/mL]@48 hrs

% inhibition   

yield [10000/mL]@72 hrs

 % inhibition
 Control  1.014  -  7.821    50.941  -
 2.56  1.228  -21.1  8.754  -11.9  56.959  -11.8
 6.4  1.157  -14.1  8.324  -6.4  53.202  -4.4
 16  1.228  -21.1  7.893  -0.9  42.163  17.2
 40  1.085  -7.0  5.238  33.0  7.587  85.1
 100  0.8  21.1  1.865  76.1  1.991  96.1

Table 2: Growth rates µ and percentage inhibition of µ during test period

 Loading rate and dilutions

[mg test item/L]

growth rates µ

0 -24 hrs 

 % inhibition

0 -24 hrs

 growth rates µ

0 -48 hrs

 % inhibition

0 -48 hrs

growth rates µ

0 -72 hrs 

% inhibition

0- 72 hrs

 
control   1.106  -  1.405  -  1.544  -  
 2.56  1.238  -12.0  1.459  -3.9  1.581  -2.4  
 6.40  1.196  -8.2  1.435  -2.2  1.558  -1.0  
 16  1.238  -12.0  1.410  -0.4  1.482  4.0  
 40  1.154  -4.4  1.220  13.1  0.927  39.9  
 100  0.953  13.8  0.777  44.7  0.534  65.4  

Microscopic examination of  algal cells: Shape of algal cells after 72 hours exposure time versus control items did not show any difference for up to 16 mg/L nominal concentration of test item. For 40 and 100 mg test item/L cells were observed to be smaller but thicker.

Study Design

The objective of this study was to determine the influence of the test item on the growth and growth rate of the freshwater green algae species Pseudokirchneriella subcapitata. The study design included one control group with six replicates and five test item groups with three replicates, each containing 50 mL reconstituted water or test medium and about 5000 cells/mL at the start of the experimental phase. The study was performed with nominal concentrations of 2.56, 6.4, 16, 40 and 100.0 mg/L in reconstituted water. The growth of the algae was calculated after 24, 48, and 72 hours exposure in the test item and control groups. Analytical controls of the test item preparation were performed at the start of the study and after 72 hours.

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 201.

Results

 Parameter Refering to

72 hours exposure time

 Yield

[mg test item/L]

 Growth rate

[mg test item/L]

 EC50

 24.8 (95% CI: 23.7 - 25.9)

 60.9 (95% CI: 59.3 - 62.7)

 EC20

 16.8 (95% CI: 15.8 -17.7)

 25.6 (95% CI: 24.5 -26.7)

 EC10

 13.7 (95% CI: 12.6 - 14.6)

 16.3 (95% CI: 15.3 -17.2)

 NOEC

 6.4

 6.4

 LOEC  16  16

Conclusion

The test material, solved in reconstituted water, was tested in a static concentration response test with 5 different loadings.  Under the conditions of the present study, the 72h EC50 value was 24.8 mg test item/L (yield) and 60.9 mg test item/L  for the growth rate

Validity criteria fulfilled:
yes
Conclusions:
The test material, solved in reconstituted water, was tested in an static concentration response test with 5 different loadings.
Under the conditions of the present study, the 72h ErC50 value was 60.9 mg test item/L (for the growth rate). ErC10 values were 16.3 mg test item/L for growth rate.
Highest nominal concentration without statistical significant effects (NOEC) was 6.4 mg test item/L.
Lowest nominal concentration with statistical significant effects (LOEC) was 16 mg test item/L.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
For this endpoint information from a structural similar compound is available. This study for this similar compound was performed according to GLP and the methods applied are fully compliant with OECD TG 201. See chapter 13 report for a more detailed justification.
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
60.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CI 95% 59.3-62.7
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
16.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CI 95% 15.3-17.2
Validity criteria fulfilled:
yes

Description of key information

For a structural analogue substance, the follwoing values were determined based on growth rate.

ErC50        60.9 mg/L

ErC20        25.6 mg/L

ErC10        16.3 mg/L

NOEC       6.4 mg/L

LOEC       16 mg/L

Key value for chemical safety assessment

EC50/LC50 for freshwater algae:
60.9 mg/L
EC10, LC10 or NOEC for freshwater algae:
16.3 mg/L

Additional information

The structural analogue substance solved in reconstituted water, was tested in an static concentration response test with 5 different loadings.   The concentrations of the test item were determined in the test media samples from all test concentrations and the control samples from all sampling times. Two of the three components were analysed. Due to limited analytical techniques and the low content of Europium only Barium and Strontium were measured. The concentrations of both measured components were within ± 20% of the measured initial concentrations during the test. Therefore, the test item was stable in the test media during the test duration. The measured concentrations show that the lower test concentrations were prepared correctly.

Under the conditions of the present study, the 72h ErC50 value was 60.9 mg test item/L  for the growth rate. ErC20 values were and 25.6 mg test item/L (growth rate). ErC10 values were 16.3 mg test item/L for growth rate. Highest nominal concentration without statistical significant effects was 6.4 mg test item/L.  Lowest nominal concentration with statistical significant effects was 16 mg test item/L.