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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
effects on growth of green algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Jan 2022 to 21 Mar 2022
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Lot No.: 108404E
Appearance: White powder
Expiry date: August 03, 2022
Storage: Room temperature, protected from light
Analytical monitoring:
yes
Details on sampling:
The test item content of the test solutions was measured at the start and at the end of the test.
After dilution the samples were analysed by HPLC method.
For determination of the test item concentrations in the test solutions, all three test vessels
were analysed at the start and at the end of the test. In case of the control samples three test
vessels were analysed at the start and at the end of the test.
For concentrations see any other information on materials.
Vehicle:
no
Details on test solutions:
The test item was completely and evenly suspended in OECD medium by mechanical
dispersion without use of any solubilising agent in the testing laboratory (Validation of the
Analytical Method for the Determination of SBCAT-03 for the Eco-toxicological Studies
performed by TOXI-COOP ZRT., Study No.: 950-100-6322).
0.0114 g of test item was suspended in 1140 mL of the dilution water (OECD Medium; see
section 5.4) in order to give the nominal loading rate of 10 mg/L of the test item and handled
in ultrasonic bath for 60 minutes. Thereafter, this stock solution was filtrated through a
membrane filter (0.45 μm)* to separate the possible non-dissolved test material. The further
test item concentrations were prepared from this test item stock solution (TISS) as shown in any other information on materials. After the formulation procedure algal cells were immediately introduced into the test
solutions.
*Type: Thermo NALGENE®
Lot number: 1264247
Expiration date: September 2024
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Raphidocelis subcapitata
(also known as Pseudokirchneriella subcapitata and Selenastrum
capricornutum)
Strain number: 61.81 SAG
(identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Origin: The algae were supplied by the SAG: Collection of Algal Cultures,
Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2,
D-37073 Göttingen, Germany
Breeding Conditions: The stock cultures are small algal cultures that are planted on agar
regularly. These are transferred to fresh medium at least once every
two months under standardized conditions according to the test
guidelines.
Pre-culturing: The pre-culture is intended to give a number of algae suitable for the
inoculation of test cultures. The pre-culture was prepared with
OECD Medium, incubated under the conditions of the test and used
when still exponentially growing, normally after an incubation
period of 2-4 days. (The pre-culture was incubated for four days
before this test.) The algal cultures used in this study did not contain
deformed or abnormal cells.
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
no details in study report
Test temperature:
The temperature was between 22.4 and 22.9°C measured in the flask and in the range of 22.0 – 23.6°C measured within the climate chamber.
pH:
The pH was measured in each test group at the start (before test solutions had been added into the test vessels) and in each test vessel at the end of the test. The pH of the control medium did not increase by more than 1.5 units during the test. The range of the pH was 7.12 – 8.22 during the experiment. The pH of the test solutions was not adjusted during the experiment.
Dissolved oxygen:
no details in study report
Salinity:
no details in study report
Conductivity:
no details in study report
Nominal and measured concentrations:
See any other information on materials
Details on test conditions:
Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water in the experiment.
Separate stock solutions were first prepared in deionised water (prepared in TOXI-COOP ZRT. by by RiOs 5 Smart water purification system, Serial No.: F7CA82540B). The growth medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations in the prepared algal growth medium.

The test containers were kept during the test in a climate chamber with controlled environmental conditions.
Temperature: Culture temperature was checked at the beginning of the test and every 24 hours thereafter in a flask filled with water, in the climatic chamber. In addition, ambient temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was between 22.4 and 22.9°C measured in the flask and in the range of 22.0 – 23.6°C measured within the climate chamber.
pH: The pH was measured in each test group at the start (before test solutions had been added into the test vessels) and in each test vessel at the end of the test. The pH of the control medium did not increase by more than 1.5 units during the test. The range of the pH was 7.12 – 8.22 during the experiment. The pH of the test solutions was not adjusted during the experiment.

Light Intensity: The light intensity was checked and recorded at the start of the test. The algal culture flasks were continuously illuminated. The average light intensity measured at the position occupied by algal culture flasks at the start of the test was 7322 lux (SD: 162 lux), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.
Reference substance (positive control):
yes
Remarks:
For the evaluation of the reliability of the applied test system and the experimental conditions Potassium dichromate is tested at least twice a year.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.571 mg/L
95% CI:
> 0.486 - < 0.666
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.827 mg/L
95% CI:
> 1.644 - < 2.027
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.62 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and yield
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.203 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and yield
Details on results:
The cell density in the untreated control cultures increased exponentially by a factor of
73.00 within 72 hours. This corresponds to a specific growth rate of 1.43 day-1.
The mean coefficient of variation (CV) for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72 h-tests) in the control cultures was 26.38 %, thus not exceeding the validity criterium of 35 %.
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the replicate control cultures was 2.21%, thus not exceeding the validity criterium of 7 %.
All validity criteria were met, therefore the study is considered as fully valid.
10.2 Analytical Results
Six different test concentrations arranged in a geometric series with a spacing factor of 2.5 and a concurrent control was tested in the main experiment. The concentrations of the test item were analysed at the start and at the end of the test.
No interfering component was detected in the untreated control samples. The mean of measured test item concentrations were in the range of 64 - 109 % of the nominal concentrations during the experiment, therefore the biological results are based on the measured and calculated test item concentrations.
A summary of the measured and calculated test item concentrations is given in any other information on materials.

10.3 Biological Results

The analytically measured test item concentrations did not remain within ± 20 % of the nominals by the end of the experiment at the two lowest test concentrations, therefore the biological results are based on the geometric mean of the measured concentrations.

10.3.1 Average Specific Growth Rate and Yield

The results of the statistical evaluation (Multiple Sequentially-rejective Welsh-t-Test after Bonferroni-Holm, α = 0.05) showed that the 0-72 h average specific growth rate and yield was statistically significantly different from the untreated control value at the test concentrations of 0.62, 1.6, 4 and 10 mg/L.
Accordingly, the 72-h NOEC based on growth rate and yield was determined to be
0.203 mg/L, while the 72-h LOEC based on growth rate and yield was determined to be
0.62 mg/L.
The 72-h ErC50 value was determined to be 1.8265 mg/L (95 % confidence limits: 1.6442 – 2.0274 mg/L), (Probit analysis).
The 72-h EyC50 value was determined to be 0.5710 mg/L (95 % confidence limits: 0.4858 – 0.6663 mg/L), (Probit analysis).

10.3.2 Morphological Changes of Algal Cells

No abnormal shape of algal cells was noticed in the control group and at the test item concentration of 0.0765 (nominal 0.1024) mg/L during the experiment. As sign of toxicity smaller cells were found in the test groups of 0.203 (nominal 0.256), 0.62 (nominal 0.64), 1.6, 4 and 10 mg test item/L.

Results with reference substance (positive control):
For the evaluation of the reliability of the applied test system and the experimental conditions Potassium dichromate is tested at least twice a year.
The date of the latest study performed in the laboratory of TOXI-COOP ZRT. (Study number: 392-201-6326) with the reference item Potassium dichromate (K2Cr2O7) was: 21 – 24 September 2021, Final Report issued: 15 November 2021.
The 72h ErC50: 0.90 mg/L, (95 % confidence limits: 0.81 – 1.00 mg/L)
The 72h EyC50: 0.34 mg/L, (95 % confidence limits: 0.29 – 0.38 mg/L)
Reported statistics and error estimates:
Mean values and standard deviations were calculated for the test groups at each observation period using Microsoft Excel for Windows software.
For the determination of the LOEC and NOEC, the calculated mean growth rates and yield at the test concentrations were tested on significant differences to the control values using Welsh-t-Test with Bonferroni-Holm-adjustment (α = 0.05), by statistical software program ToxRatPro Version 3.3.0). The 72-hour EC10, EC20, EC50 values and their 95 % confidence limits for growth rates and yield were calculated by Probit analysis (statistical software program ToxRatPro Version 3.3.0). Details of statistical evaluation are shown in "illustration"

Summary of the Biological Endpoints

















































Endpoints


(0-72 h)


 



Growth rate (mg/l)


 



Yield


(mg/l)



Calculation based on measured concentrations


 



EC10


95 % conf. limits



0.4351


0.3377 – 0.5309



0.1827


0.1202 – 0.2394



EC20



0.7119



0.2701



95 % conf. limits



0.5906 – 0.8280



0.1983 – 0.3331



EC50



1.8265



0.5710



95 % conf. limits



1.6442 – 2.0274



0.4858 – 0.6663



NOEC



0.203



0.203



LOEC



0.62



0.62


Validity criteria fulfilled:
yes
Conclusions:
In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item SBCAT-03 had inhibitory effects on the growth of Raphidocelis subcapitata.

All validity criteria were met and therefore the study is considered as fully valid.

Biological results and endpoints are based on the measured and calculated test item concentrations.

Biological endpoints of the study are summarised in any other information on results
Executive summary:

Test item:            SBCAT-03 (Lot No.: 108404E)


Test species: Raphidocelis subcapitata (also known as Pseudokirchneriella subcapitata); Strain number: 61.81 SAG


Exponentially-growing cultures; preculture was incubated for four days before the test.


Dilution water:  OECD medium, prepared in the laboratory of TOXI-COOP ZRT.


Concentrations: Nominal concentrations of 0.1024, 0.256, 0.64, 1.6, 4 and 10 mg test item / L were investigated in the main study; a concurrent control was investigated in parallel.


Mean of the measured test item concentrations did not remain within


± 20 % of the nominal by the end of the experiment in the two lowest test concentrations, therefore the biological results are based on the geometric mean of the measured concentrations. The corresponding calculated test item concentrations were 0.0765, 0.203, 0.62, 1.6, 4 and 10 mg test item/L.


Test design:   Exponentially-growing cultures of Raphidocelis subcapitata were exposed to the test item under defined conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included three replicates at the test concentrations and six replicates for the untreated control. The algae cell concentration was approximately 104 cells/mL at the start of the test in all of the test cultures. Glass Erlenmeyer flasks with total capacity of 250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The algae cell concentration was determined by manual cell counting using microscope in each testing flask during the 72-hour test, in 24-hour intervals.


Analytics:           For determination of the test item concentrations, samples were taken from the applied concentration levels and control at the start and at the end of the test. Concentrations were determined using an HPLC method.


Endpoints:           72-hour EC10, EC20, EC50, NOEC and LOEC for both response variables (i.e. average specific growth rate and yield).


Statistics:            For the determination of the LOEC and NOEC, Welsh-t-Test with Bonferroni-Holm-adjustment (a = 0.05) was used (statistical software program ToxRatPro Version 3.3.0). The EC10, EC20, EC50 values were calculated by Probit analysis (statistical software program ToxRatPro Version 3.3.0).


Validity:              All validity criteria were met and therefore the study is considered as fully valid (see section 10.1)


Results:               All biological results are based on the geometric mean of the measured test item concentrations. The biological endpoints are summarised below in Table 1.


Table 1: Summary of the Biological Endpoints

















































Endpoints


(0-72 h)


 



Growth rate (mg/l)


 



Yield


(mg/l)



Calculation based on measured concentrations


 



EC10


95 % conf. limits



0.4351


0.3377 – 0.5309



0.1827


0.1202 – 0.2394



EC20



0.7119



0.2701



95 % conf. limits



0.5906 – 0.8280



0.1983 – 0.3331



EC50



1.8265



0.5710



95 % conf. limits



1.6442 – 2.0274



0.4858 – 0.6663



NOEC



0.203



0.203



LOEC



0.62



0.62



 


 


 

Description of key information

In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results
showed that the test item SBCAT-03 had inhibitory effects on the growth of Raphidocelis
subcapitata.
All validity criteria were met and therefore the study is considered as fully valid.
Biological results and endpoints are based on the measured and calculated test item
concentrations.
Biological endpoints of the study are summarised below:

















































Endpoints


(0-72 h)


 



Growth rate (mg/l)


 



Yield


(mg/l)



Calculation based on measured concentrations


 



EC10


95 % conf. limits



0.4351


0.3377 – 0.5309



0.1827


0.1202 – 0.2394



EC20



0.7119



0.2701



95 % conf. limits



0.5906 – 0.8280



0.1983 – 0.3331



EC50



1.8265



0.5710



95 % conf. limits



1.6442 – 2.0274



0.4858 – 0.6663



NOEC



0.203



0.203



LOEC



0.62



0.62


Key value for chemical safety assessment

EC50 for freshwater algae:
1.827 mg/L

Additional information