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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:
From 2017-05-08 to 2017-05-12, with the definitive exposure phase from 2017-05-09 to 2017-05-12
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
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures. OECD series on testing and assessment no. 23, ENV/JM/MONO(2000)6
Deviations:
not applicable
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Type of analysis and analysis dates
The limit concentration and the control were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) at the start and at the end of the exposure. Separate replicates for the test item analysis after 0 and 72 hours were prepared without algae and incubated under test conditions.
Vehicle:
no
Details on test solutions:
Water Accommodated Fraction (WAF)
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000) is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents. Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. After completion of mixing and following a settlement period, the test item phase was separated by siphon and the test organisms were exposed to the aqueous phase, the WAF (which may contain dissolved and/or suspended and/or emulsified fractions of the test item mixture). Exposure was expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of the test item in the WAF.

Preparation of the water accommodated fraction
One water accommodated fraction (WAF) was prepared with a nominal loading of the test item of 100 mg/L. The loading level is based on the results of a preliminary range finding test (non GLP, open system).
An appropriate amount of the test item was weighed out onto a glass slide. The glass slide with the test item was inserted in a brown glass flask with an appropriate amount of demineralized water. These dispersion was shaken for 24 hours with 20 rpm at room temperature. After a separation phase of 1 hour, the WAF was removed by siphoning (from the approximate centre of the flask) and used in the test. Thereafter, the components of the dilution water were added to the WAF. The WAF was checked via laser beam for undissolved test item, which was positive.

Test loading
Per definition of the WAF, all terms related to concentration level were given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations. The WAF was tested in a limit test.

Control
Six replicates (without test item) were tested under the same test conditions as the test item replicates. The dilution water for the preparation of the control was treated as the same as the WAF.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.


Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
No
Hardness:
Not specified
Test temperature:
min.: 21.8 max.: 23.0 mean value: 22.4
pH:

Nominal loading level pH-value [mg/L] Start; 0 hours End; 72 hours
100 8.04 9.27
Control 8.02 9.41

Dissolved oxygen:
Not measured
Salinity:
Not measured, freshwater conditions
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 10^3 cells/mL, Actual: mean 6839 cells/mL
- Control end cells density: Mean 2570048 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2



OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120
µE ∙ m-2 ∙ s-1, mean value: 5056 lux,



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
Cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the preliminary range finding test.


Reference substance (positive control):
yes
Remarks:
(Potassium dichromate)
Key result
Duration:
72 h
Dose descriptor:
other: NOEL
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: LOEL
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: NOEL
Effect conc.:
< 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
other: LOEL
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
< 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: The test item concentrations were clear during the exposure. The algae in the saturated solution were agglutinated after 72 hours. No agglutination of the algae was observed in the control replicates.
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
The toxicity of potassium dichromate (SIGMA, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2016-10-11 to 2016-10-14 (with headspace). The reference item toxicity is in the valid range following test facility SOPS.

EC50-Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.435 0.764 ± 0.513
95 % confidence interval 0.414 – 0.458
Yield inhibition
EyC50 0.230 0.402 ± 0.293
95 % confidence interval 0.179 – 0.322
SD = Standard deviation

Reported statistics and error estimates:
EL-values and statistica analyses
EL-values of the growth rate and yield inhibition were estimated empirically based on the results of the only treatment level (Limit test design).

NOEL and LOEL values
NOEL/LOEL were determined by calculation of statistical significance of growth rate and yield. As a standard, a t-test was used for NOEL/LOEL calculations. When running a t-test a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normal distribution of data. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

Software
The data for the tables in this report are computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
•Excel, MICROSOFT CORPORATION
•SigmaPlot, SPSS INC.

Summary tables on results

Cell Densities

Nominal loading level

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

100

1

6839

39519

301674

2202299

2

6839

40865

284025

2101937

3

6839

41021

343423

2053248

4

6839

41076

321911

2050030

5

6839

38080

343189

2484775

6

6839

41717

348912

2211955

Mean

6839

40380

323856

2184041

Control

1

6839

38984

425503

2901847

2

6839

42118

368153

2649860

3

6839

40137

456355

2437900

4

6839

37430

377996

2773103

5

6839

36719

358544

2344210

6

6839

35932

353945

2313370

Mean

6839

38553

390083

2570048

 

 

 Evaluation after 72 hours

 Statistically significant differences of growth rates and yield compared to
control values are marked (+), not significant differences are marked (-).

Nominal loading level

Replicate

Growth rate

Growth rate inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

100

1

 

1.93

3

 

2195460

14

2

                                                                                        

1.91

3

 

2095098

18

3

 

1.90

4

 

2046409

20

4

 

1.90

4

 

2043191

20

5

 

1.97

1

 

2477936

3

6

 

1.93

2

 

2205116

14

Mean

(+)*

1.92

3

(+)

2177202

15

Control

1

 

2.02

 

 

2895008

 

2

 

1.99

 

 

2643021

 

3

 

1.96

 

 

2431061

 

4

 

2.00

 

 

2766264

 

5

 

1.95

 

 

2337371

 

6

 

1.94

 

 

2306531

 

Mean

 

1.98

 

 

2563209

 

* = statistically significant, but biologically not relevant (inhibition < 5%)

 

Section-by-Section and Average Specific Growth Ratesof the Control Group (0 - 72 hours)

 

Replicate No.

Specific growth rate [d-1]

Mean

(0 - 72 hours)

SD

±

CV
[%]

Mean CV [%]

section-by-section

0 - 24 hours

24 - 48 hours

48 - 72 hours

Control

1

1.74

2.39

1.92

2.02

0.336

16.6

15.6

2

1.82

2.17

1.97

1.99

0.175

8.8

3

1.77

2.43

1.68

1.96

0.412

21.0

4

1.70

2.31

1.99

2.00

0.306

15.3

5

1.68

2.28

1.88

1.95

0.305

15.7

6

1.66

2.29

1.88

1.94

0.319

16.4

 

 

 

Mean

1.98

 

 

 

 

 

SD ±

0.03

 

 

 

 

CV [%]

1.58

 

SD = Standard deviation CV = Coefficient of variation

Validity criteria fulfilled:
yes
Conclusions:
In this study, no inhibition of the growth rate but an effect on yield on the freshwater green alga Pseudokirchneriella subcapitata was found after 72 hours exposure to the test item WARADUR® LG. The nominal EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 100 mg test item/L, respectively. The nominal NOEC-value for inhibition of growth rate was 100 mg test item/L. The NOEC-value for yield was < 100 mg test item/L.
All effect values given are based on nominal loading level of WARADUR® LG.
Executive summary:

The toxicity of WARADUR® LG (batch number: W00004534) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/ Method C.3 from 2017-05-08 to 2017-05-12, with the definitive exposure phase from 2017-05-09 to 2017-05-12 at the test facility. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

 

The study was conducted under static conditions with an initial cell density of 6839 cells/mL. A WAF with a nominal loading level of 100 mg test item/L was tested. Six replicates were used for the limit concentration and the control. The environmental conditions were within the acceptable limits.

 

The test media were clear throughout the test period. The concentrations of the test item were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) at the start of the exposure (fresh media, 0 hours) and at the end of the exposure (old media, 72 hours).

All effect values given are based on the nominal loading level of WARADUR®LG.

 

 NOEC, LOEC, EC - values of WARADUR®LG (0-72hours)

                  based on the nominal loading level [mg/L]

 

 

Inhibition of Growth Rate

NOEC

  100

LOEC

> 100

ErC10

> 100

ErC20

> 100

ErC50

> 100

 

Inhibition of Yield

NOEC

< 100

LOEC

  100

EyC10

< 100

EyC20

> 100

EyC50

> 100

Description of key information

In eight reliable guideline studies (reliability category 1) with different members of the Montan waxes category no signs of toxicity were observed using saturated solutions prepared at nominal loading levels between 100 to 320 mg/L.

EC10/LC10 or NOEC for freshwater algae (growth rate) > water solubility limit (OECD 201, 2009 / 2012 / 2017)

Key value for chemical safety assessment

Additional information

Please refer to CSR for further details on study summaries of different members of the Montan waxes category as well as Read-across justification in section 13 'assessment reports'. 

Montan waxes, types LG, KPS, WM 31, O, NaV 101, S, E, and OP were tested in reliable (reliability category 1) OECD 201 tests with the unicellular freshwater green alga Desmodesmus subspicatus or Pseudokirchneriella subcapitata. All studies were performed compliant to GLP and meeting the validity criteria of the OECD guideline. Static test design was applied. Due to the poor solubility of the test items, water accommodated fractions (WAF) were tested prepared at the nominal limit concentration of 100 mg/L (all but type E) or loading levels up to 320 mg/L (type E). Poor water solubility of the Montan waxes precluded their analytical quantification in the saturated solutions. No inhibition of growth was observed with any of the Montan waxes types tested at nominal concentrations well above their solubility limits. Minor effects on yield of biomass were observed for type LG. In summary, Montan waxes category members are concluded to be acutely and chronically non-toxic to aquatic plants / algae.

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