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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:
2012-11-23 to 2012-11-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
(2006)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Various concentrations of the test item and the control were analytically verified after 0 hours (test start) and 72 hours (test end) via HPLC-DAD analysis. Separate replicates for the test item analysis after 0 and 72 hours were prepared (test beginning provided without alga and test end with same density of alga as the test replicates) and incubated under test conditions.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
A saturated solution was freshly prepared with dilution water. 0.5 mL /1080 mL was used as loading rate. The dispersion was slowly stirred for 72 hours (300 rpm, room temperature) with a magnetic stirrer. Thereafter the saturated solution was decanted.
The saturated solution and further 4 dilution levels were tested in a geometrical series with a dilution factor of 3.16 (nominal): 1.00, 3.16, 10.0, 31.6, 100 % of the saturated solution.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Green alga
- Strain: Desmodesmus subspicatus CHODAT SAG 86.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany
- Age of inoculum (at test initiation): three day old preculture
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity: 35 – 70 µE/m²/s for 24 h per day

ACCLIMATION
- Acclimation period: no data
- Culturing media and conditions (same as test or not): Nutrient medium Z according to LÜTTGE et al. (1994)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22-23°C
pH:
initial: 7.90-8.09
final: 7.88-9.17
Nominal and measured concentrations:
nominal: 1.00, 3.16, 10.00, 31.60, 100% saturated solution, corresponding to measured (geometric mean) 1.54, 4.95, 18.8, 60.7 and 197 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: sterile headspace flasks (volume 119 mL) with aluminium tops and PTFE seals, test volume 119 mL, with regard to the volatility of the test item, glass flasks without headspace were used to reduce the losses of the test item
- Aeration: agitation, test containers were placed on a rotary shaker and oscillated at approximately 70 rpm
- Initial cells density: 4332 cells/mL
- Control end cells density: 270696 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to OECD guideline 201

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24 h light
- Light intensity and quality: 60 – 120 µE/m²/s (measured 98.3 µE/m²/s, CV 5.38%)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Cell density was measured daily via Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.6
- Range finding study
- Test concentrations: 0.1, 1.0, 10.0, 100.0% saturated solution
- Results used to determine the conditions for the definitive study: growth rate inhibition, 0% at 0.1% saturated solution, 0% at 1.0% saturated solution, 13% at 10% saturated solution and 80% at 100% saturated solution
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
18.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
23.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 18.4-33.7 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
39.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 32.2 - 45.1 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.54 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 95% CL <1.54 - 4.39 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
29.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 95% CL 23.4 - 42.3 mg/L
Details on results:
- Observation of abnormalities (for algal test): microscopic evaluation of the cells at start and end of the incubation period revealed no morphological abnormalities
Results with reference substance (positive control):
- Results with reference substance valid? yes
- ErC50: 0.631 mg/L, 95% c.i. 0.583 - 0.687 mg/L; valid range: 0.682 ± 0.452 mg/L
- EyC50: 0.256 mg/L, 95% c.i. 0.247 - 0.265; valid range: 0.314 ± 0.144 mg/L

Mean cell densities [cells/mL] during the final test:

Geometric mean measured test item concentration[mg/L]

Exposure time [h]

0

24

48

72

197

4332

4466

<LOQ

<LOQ

60.7

4332

9482

9724

7019

18.8

4332

13594

51400

212025

4.95

4332

12106

44950

195650

1.54

4332

16534

66489

279842

Control

4332

14821

64637

275028

  LOQ = Limit of quantification of fluorescence intensity of Desmodesmus subspicatus (2280 cells/mL)

 

Growth rate, yield during the final test: 

Geometric mean measured test item concentration [mg/L]

Growth rate [/d]

Rate-related inhibition [%]

Yield [cells/mL]

Inhibition of yield [%]

197

0 (+)

100

0 (+)

100

60.7

0.16 (+)

88.4

2687 (+)

99

18.8

1.30 (+)*

6.13

207693 (+)

23.3

4.95

1.27 (+)*

8.06

191318 (+)

29.3

1.54

1.39 (-)

-0.57

275510 (-)

-1.78

control

1.38

270696

 

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

*) biologically not significant (inhibition< 10 %)

 

The study meets the validity criteria of the guideline:

The cell growth increased 63-fold after 72 h in the control (required: at least 16-fold).

The mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures was 13.9% (required: < 35%).

Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 3.04% (required: < 7%).

Validity criteria fulfilled:
yes
Conclusions:
In this study TFMEA was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours with the following effect values.
The NOEC-values for inhibition of growth rate and yield after 72 hours were 18.8 and 1.54 mg/L, respectively.
The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) with 95% confidence intervals after 72 hours were 23.1 (18.4-33.7) and 2.14 (<1.54 - 4.39) mg/L, respectively.
The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) with 95% confidence intervals after 72 hours were 39.5 (32.2 - 45.1) and 29.9 (23.4 - 42.3) mg/L, respectively.
All effect levels are given based on geometric mean measured concentrations of TFMEA.
Executive summary:

In a 72 hour acute toxicity study, the cultures of Desmodesmus subspicatus CHODAT SAG 86.81 were exposed to TFMEA at concentrations of nominal 1.00, 3.16, 10.00, 31.60, 100% saturated solution, corresponding to measured (geometric mean) 1.54, 4.95, 18.8, 60.7 and 197 mg/L under static conditions in accordance with the OECD guideline 201 (adopted 23 March 2006). 

The NOEC-values for inhibition of growth rate and yield after 72 hours were 18.8 and 1.54 mg/L, respectively.

The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) with 95% confidence intervals after 72 hours were 23.1 (18.4-33.7) and 2.14 (<1.54 - 4.39) mg/L, respectively.

The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) with 95% confidence intervals after 72 hours were 39.5 (32.2 - 45.1) and 29.9 (23.4 - 42.3) mg/L, respectively.

Microscopic evaluation of the cells at start and end of the incubation period revealed no morphological abnormalities.

All effect levels are given based on geometric mean measured concentrations of TFMEA.

 

Results Synopsis

 

Test Organism: Desmodesmus subspicatus CHODAT SAG 86.81

Test Type: Static

 

72 hr ErC50: 39.5mg/L              95% C.I.: 32.2 - 45.1mg/L

72 hr EyC50: 29.9mg/L             95% C.I.: 23.4 - 42.3mg/L

72 hr ErC10: 23.1 mg/L              95% C.I.: 18.4-33.7mg/L

72 hr EyC10: 2.14 mg/L             95% C.I.: <1.54 - 4.39mg/L

72 hr NOEC(growth rate): 18.8 mg/L              

72 hr NOEC(yield): 1.54 mg/L              

 

Endpoint(s) Effected:  growth rate, yield

Description of key information

72 h ECr10 = 23.1 mg/L (95% c.i. 18.4 - 33.7 mg/L) / 72 h ErC50 = 39.5 mg/L (95% c.i. 32.2 - 45.1 mg/L); OECD guideline 201; GLP

Key value for chemical safety assessment

EC50 for freshwater algae:
39.5 mg/L
EC10 or NOEC for freshwater algae:
23.1 mg/L

Additional information

One short-term study in freshwater algae is available for TFMEA, which is considered reliable without restriction (Klimisch score 1).

In a 72 hour acute toxicity study, the cultures of Desmodesmus subspicatus CHODAT SAG 86.81 were exposed to TFMEA at concentrations of nominal 1.00, 3.16, 10.00, 31.60, 100% saturated solution, corresponding to measured (geometric mean) 1.54, 4.95, 18.8, 60.7 and 197 mg/L under static conditions in accordance with the OECD guideline 201 (adopted 23 March 2006).  

Microscopic evaluation of the cells at start and end of the incubation period revealed no morphological abnormalities.

The NOEC-values for inhibition of growth rate and yield after 72 hours were 18.8 and 1.54 mg/L, respectively.

The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) with 95% confidence intervals after 72 hours were 23.1 (18.4-33.7) and 2.14 (<1.54 - 4.39) mg/L, respectively.

The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) with 95% confidence intervals after 72 hours were 39.5 (32.2 - 45.1) and 29.9 (23.4 - 42.3) mg/L, respectively. All effect levels are given based on geometric mean measured concentrations of TFMEA.