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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:
2020-01-15 to 2020-01-20
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:
adopted on 23 March 2006, corrected on 28 July 2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment. Additional duplicate samples were taken after 24 hours and 48 hours from additionally prepared vessels from each test item concentration and the control prepared for this purpose.
The sample volume was 10 mL.
- Sample storage conditions before analysis: All samples were extracted with dichloromethane immediately after sampling due to the risk of degradation of the test item in the aqueous samples during storage. The organic solvent extracts were stored deep frozen (≤ -20 °C) until analysis.
Vehicle:
no
Details on test solutions:
Due to the low limit of water solubility of the test item, a Water Soluble Fraction (WSF) of nominal 100 mg/L and dilutions of 1:3.2, 1:10.24, 1:32.8 and 1:104.9 of this WSF were tested.
The highest test item concentration of 100 mg test item/L was prepared by mixing 99.5 mg test item into 995 mL test water (Reconstituted Water OECD Medium) and carefully stirred for 1 hour at room temperature in the dark to dissolve as much of the test item as possible. After cessation of mixing and a following period (40 min) of settling to allow phase separation, the aqueous phase, i.e. the water soluble fraction, was drawn off carefully and used as the test medium of the highest nominal test concentration of 100 mg test item/L. Adequate volumes of this water accommodated fraction were mixed into test water to obtain the desired dilutions of 1:3.2, 1:10.24, 1:32.8 and 1:104.9.
The test media were prepared just before introduction of the algae (= start of the test).
In the control, test water was used without addition of the test item.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular green algae
- Strain: 61.81 SAG
- Source: „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
- Cultivation: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg/L as CaCO3 (calculated)
Test temperature:
21.4 to 22.1 °C
pH:
Control: 8.0 (0h) to 9.0 (72h), Test item treatments: 8.0 (0h) to 8.8 – 9.2 (72h)
Nominal and measured concentrations:
Nominal: Water Soluble Fraction (WSF) of nominal 100 mg/L and dilutions of 1:3.2, 1:10.24, 1:32.8 and 1:104.9 of this WSF,
Geometric mean measured: 0.0 (control), 7.28, 8.40, 10.3, 18.6 and 249 µg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks of 50 mL volume with nominal 50 mL of test medium loosely covered with watch glass
- Type: closed
- Initial cells density: Nominal 5000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water according to OECD TG 201
- Intervals of water quality measurement: The pH was measured in all test item concentrations and the control at the start and the end of the test. The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media.
- Photoperiod: 24 hours/day light
- Mean light intensity: 4983 lux (range: 4800 to 5160 lux)

EFFECT PARAMETERS MEASURED:
- Determination of cell density: spectrophotometric measurement

TEST CONCENTRATIONS
- Range finding study: yes
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
18.6 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 249 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Confidence interval not determinable
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
169.6 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
yield
Remarks on result:
other: 95 % confidence interval: 97.8 - > 249
Details on results:
The 72-hour EyC50 was calculated to be 169.6 μg test item/L and the ErC50 was calculated to be > 249 μg test item/L based on the geometric mean concentrations. Thus, the ErC50 value was higher than the saturated solution of the test item in this study. The 72-hour NOEC for yield and growth rate was determined to be 18.6 μg test item/L and the associated 72-hour LOEC for both, yield and growth rate, was calculated to be 249 μg test item/L.
Results with reference substance (positive control):
Results of the most recent test with Pseudokirchneriella subcapitata performed with the reference item Potassium Dichromate in July 2019:
72-hour ErC50 (95 % conf. interval): 1.34 mg/L (1.29 – 1.39 mg/L)
72-hour ErC20 (95 % conf. interval): 0.631 mg/L (0.592 – 0.669 mg/L)
72-hour ErC10 (95 % conf. interval): 0.426 mg/L (0.390 – 0.461 mg/L)
72-hour NOEC: 0.20 mg/L
72-hour LOEC: 0.63 mg/L

Biological results


Table 1. Growth rate during the test period of 72 hours




























































Geometric mean measured concentration



Flask



Growth rates μ [1/day] after



[μg test item/L]



No.



0 - 24 hours



0 - 48 hours



0 - 72 hours



Control



1


2


3


4


5


6


m


s



1.533


1.597


1.597


1.657


1.533


1.657


1.595


0.056



1.583


1.726


1.627


1.716


1.690


1.764


1.684


0.067



1.625


1.720


1.645


1.702


1.647


1.696


1.673


0.038



7.28



1


2


3


m


s



1.657


1.657


1.713


1.676


0.033



1.662


1.657


1.662


1.660


0.003



1.698


1.668


1.659


1.675


0.021



8.40



1


2


3


m


s



1.657


1.657


1.597


1.637


0.035



1.768


1.684


1.705


1.719


0.044



1.746


1.642


1.660


1.682


0.056



10.3



1


2


3


m


s



1.657


1.713


1.713


1.695


0.033



1.662


1.726


1.684


1.691


0.032



1.684


1.728


1.697


1.703


0.023



18.6



1


2


3


m


s



1.713


1.464


1.464


1.547


0.144



1.668


1.633


1.615


1.639


0.027



1.630


1.655


1.652


1.646


0.014



249



1


2


3


m


s



1.391


1.311


1.391


1.364


0.046



1.249


1.236


1.403


1.296


0.093



1.274


1.314


1.488


1.359


0.114



m: mean value; s: standard deviation


 


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


































































































 



Growth rates μ [1/day] and % inhibition of μ



Geometric mean


measured concentration



0 - 24 hours



 



 



0 - 48 hours



 



 



0 - 72 hours



 



[μg test item/L]



μ



%



 



μ



%



 



μ



%



Control



1.595



-



 



1.684



-



 



1.673



-



7.28



1.676



-5.0



 



1.660



1.4



 



1.675



-0.1



8.40



1.637



-2.6



 



1.719



-2.1



 



1.682



-0.6



10.3



1.695



-6.2



 



1.691



-0.4



 



1.703



-1.8



18.6



1.547



3.0



 



1.639



2.7



 



1.646



1.6



249



1.364



14.5



*



1.296



23.1



*



1.359



18.8



negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control


* mean value significantly different from the control (tested with Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm (24 and 72h) and Williams t-test (48h), α = 0.05, one-sided smaller)


 


Table 3. Temperature in the test media during the test period





















Water temperature [°C]



0 hours



24 hours



48 hours



72 hours



21.7



22.1



22.1



21.4



 


Table 4. pH-Values in the test media at the start and end of the test













































Geometric mean


measured concentration



pH value



[μg test item/L]



0 hours



72 hours



Control



8.0



9.0



7.28



8.0



9.1



8.40



8.0



9.1



10.3



8.0



9.2



18.6



8.0



9.2



249



8.0



8.8



No remarkable observations were made concerning the appearance of the test item in test media. All test media were clear solutions and remained clear throughout the entire test period.


 


Analytical results


Table 5. Summary of Analytical Results









































Sample Description


[mg test item/L]



Initial Mean1


[µg test item/L]



Geometric mean1,2


[µg test item/L]



Control



n.a.



n.a.



1:104.9 Dilution



48



7.28



1:32.8 Dilution



113



8.40



1:10.24 Dilution



381



10.3



1:3.2 Dilution



997



18.6



WSF of 100 mg/L



3915



249










1 Mean value of all measured samples per treatment group


2 Calculated according to OECD Guidance Document No. 23, Annex 2


The tabulated results represent results rounded to three significant digits


WSF: water soluble fraction


n.a.: not applicable


Validity criteria fulfilled:
yes
Conclusions:
Under the given conditions of this study, the 72-h ErC50 value was determined to be > 249 μg/L (95 % CI: not determinable).
Executive summary:

The acute toxicity of the test item was investigated in a GLP-study according to OECD TG 201 using unicellular freshwater green alga Pseudokirchneriella subcapitata. For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to various concentrations (7.28, 8.40, 10.3, 18.6, and 249 µg test item/L (geometric mean measured)) of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus, over several algal generations. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium. The study design included one control and five test material groups with three replicates, each containing 50 mL reconstituted water or test medium and about 5,000 cells/mL at the start of the experimental phase. The study was performed in an open static test system. The test material concentrations were analysed at 0 (start), 24, 48 and 72 hours after start of the experimental part. Defined volumes of the algal suspensions were sampled after 24, 48 and 72 for determination of cell densities with spectrophotometric measurements. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part by means of GC-MS/MS. The limit of quantification of the analytical method was 0.01 mg/L. All reported results refer to geometric mean concentrations, since the test item concentrations were not within ± 20 % of the measured initial concentrations during the test. The effect of the test item on freshwater green alga showed the following results: The 72-hour EyC50 was calculated to be 169.6 μg test item/L and the ErC50 was calculated to be > 249 μg test item/L based on the geometric mean concentrations. Thus, the ErC50 value was higher than the saturated solution of the test item in this study. The 72-hour NOEC for yield and growth rate was determined to be 18.6 μg test item/L and the associated 72-hour LOEC for both, yield and growth rate, was calculated to be 249 μg test item/L.

Description of key information

Under the given conditions of this study, the 72-h ErC50 value was determined to be > 249 μg/L (95 % CI: not determinable) (reference 6.1.5-1).

Key value for chemical safety assessment

EC50 for freshwater algae:
249 µg/L
EC10 or NOEC for freshwater algae:
18.6 µg/L

Additional information

The acute toxicity of the test item was investigated in a GLP-study according to OECD TG 201 using unicellular freshwater green alga Pseudokirchneriella subcapitata. For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to various concentrations (7.28, 8.40, 10.3, 18.6, and 249 µg test item/L (geometric mean measured)) of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus, over several algal generations. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium. The study design included one control and five test material groups with three replicates, each containing 50 mL reconstituted water or test medium and about 5,000 cells/mL at the start of the experimental phase. The study was performed in an open static test system. The test material concentrations were analysed at 0 (start), 24, 48 and 72 hours after start of the experimental part. Defined volumes of the algal suspensions were sampled after 24, 48 and 72 for determination of cell densities with spectrophotometric measurements. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part by means of GC-MS/MS. The limit of quantification of the analytical method was 0.01 mg/L. All reported results refer to geometric mean concentrations, since the test item concentrations were not within ± 20 % of the measured initial concentrations during the test. The effect of the test item on freshwater green alga showed the following results: The 72-hour EyC50 was calculated to be 169.6 μg test item/L and the ErC50 was calculated to be > 249 μg test item/L based on the geometric mean concentrations. Thus, the ErC50 value was higher than the saturated solution of the test item in this study. The 72-hour NOEC for yield and growth rate was determined to be 18.6 μg test item/L and the associated 72-hour LOEC for both, yield and growth rate, was calculated to be 249 μg test item/L.

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