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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:
February 06 - 09, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
OECD Guideline for Testing of Chemicals 201, Sect. 2: Effects on biotic systems, freshwater alga and cyanobacteria, growth inhibition test. 23 March 2006. Annex 5, corrected 28 July, 2011
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate from May 31, 2016
Analytical monitoring:
yes
Details on sampling:
Biological Sampling:
The cell concentrations were determined via an electronic particle counter in the inoculum culture prior to the addition to the test vessels at test start and after 24, 48 and 72 h in the test cultures. Light and temperature were measured daily. The solution pH was measured in one test vessel per test concentration including the control and at the end of the test, the solution pH values were measured directly in test vessels. The test culture temperature was measured in one additional test vessel.
Analytical Sampling:
Cs and Al concentrations measured at test initiation and test termination in samples of all test solutions. For this, samples (2 x 20 mL) were taken from all five test solutions and controls at the beginning of the test prior to addition of the algae (2 x 6 samples) and immediately stabilised by adding 0.2 mL conc. HNO3 to 20 mL test media.
At the end of the growth test, samples were taken directly from representative replicates per test concentration and control (2 x 6 samples). The test solutions were stabilised by adding 0.2 mL conc. HNO3 to 20 mL test media. Samples are typically stable for at least three months. Samples measured on the day of sampling were immediately stored at 4 °C until analysis.
Vehicle:
no
Details on test solutions:
Sterilised synthetic growth medium (OECD medium) according to OECD 201 was used as growth medium. Details are given in the table given under "any other information on materials nnd methods including tables". All stock solutions and the medium were prepared with purified water processed using an ELGA „PURELAB Ultra“. The pH of the medium was obtained at equilibrium between the carbonate system of the medium and the partial pressure of CO2 in atmospheric air.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Scientific name: Raphidocelis subcapitata, Selenastraceae, Chlorophycea, Chlorophyta. (former name: Pseudokirchneriella subcapitata)
- Strain: 61.81 SAG
- Source: SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Göttingen
- Inoculum (test start): 10,000 cells/mL
- Method of cultivation: stock cultures were maintained acc. to OECD 201 and Bringmann und Kühn (1980). Prior to testing a pre-culture was established in OECD growth medium for 3 days to obtain exponentially growing algae for the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
not reported
Test temperature:
22.0 °C (required: 21 to 24 °C, controlled at ± 2°C)
pH:
Control:
7.87 (test start); mean value (n = 7) of 7.94 (test end)
Treatments:
7.84 to 7.88 (test start); 7.65 to 7.92 (test end)
Dissolved oxygen:
not reported
Salinity:
not reported
Conductivity:
not reported
Nominal and measured concentrations:
Nominal
Test item: (control), 0.31, 0.93, 2.78, 8.33 and 25.0 mg test item/L
Cesium: 0 (control), 0.18, 0.54, 1.60, 4.81 and 14.43 mg Cs/L
Alumnium: 0 (control), 0.03, 0.09, 0.28, 0.85 and 2.55 mg Al/L

Mean measured (start)
Cesium: 0.00003, 0.16, 0.45, 1.39, 4.50 and 13.2 mg Cs/L
Alumnium: 0.005, 0.01, 0.03, 0.07, 0.03 and 0.65 mg Al/L

Mean measured (end)
Cesium: 0.00003, 0.15, 0.45, 1.39, 4.38 and 13.2 mg Cs/L
Alumnium: 0.005, 0.01, 0.03, 0.07, 0.01 and 0.35 mg Al/L

Arithmetic mean (based on measured cesium concentrations and following control-normalization)
Cesium: 0 (control), 0.15, 0.45, 1.38, 4.44 and 13.20 mg Cs/L
Details on test conditions:
TEST SYSTEM
- Test vessel: conical glass flasks, sterilized
- Type: covered with air-permeable silicone-sponge caps, sterilized
- Fill volume: 250 mL
- Aeration: no
- Type of flow-through: static, continuous shaking
- Initial cells density: 10,000 cells/mL
- replicates: 4 (treatmets), 7 (controls, one control replicate broke at test end, thus the final cell number was not determined)

GROWTH MEDIUM
- Standard medium used: OECD growth medium
- water: purified water processed using an ELGA „PURELAB Ultra“.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity: 91.63 to 95.12 µE m-2 s-1
- Light qualitix: OSRAM Standard “cool white"

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- cell counting: electronic particle counter

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 1, 10 and 100 mg test item/L
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (3,5-DCP)
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4.92 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (dissolved fraction)
Remarks:
cesium
Basis for effect:
growth rate
Remarks on result:
other: 95 % cl (n=4): 4.35 - 5.43 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.18 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (dissolved fraction)
Remarks:
cesium
Basis for effect:
biomass
Remarks on result:
other: 95 % cl (n=4): 1.29 - 2.94 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
8.53 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Remarks:
CsAlF4
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.78 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Remarks:
CsAlF4
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (dissolved fraction)
Remarks:
cesium
Basis for effect:
growth rate
Remarks on result:
other: 95 % cl (n=4): 7.64 - 8.76 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
5.47 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (dissolved fraction)
Remarks:
cesium
Basis for effect:
biomass
Remarks on result:
other: 95 % cl (n=4): 4.29 - 7.13 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
14.21 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Remarks:
CsAlF4
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9.48 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Remarks:
CsAlF4
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.38 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (dissolved fraction)
Remarks:
cesium
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.44 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (dissolved fraction)
Remarks:
cesium
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.39 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Remarks:
CsAlF4
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
7.69 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Remarks:
CsAlF4
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
4.44 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (dissolved fraction)
Remarks:
cesium
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
13.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element (dissolved fraction)
Remarks:
cesium
Basis for effect:
biomass
Details on results:
Control cell number increase in 72 h was > 16 (i.e. 132.7), the mean of the control replicate CV% in the section-by-section growth rate was ≤ 35% (i.e. 2.60%) & the CV of average specific growth rate after 72 h in control replicate was ≤ 7% (i.e. 5.73%).
Test item concentrations were calculated using the endpoints in mg Cs/L and the cesium content of 57.7 % in the test item.
The NOEC for growth rate was determined with 1.38 mg Cs/L based on mean measured Cs concentrations. However, due to an inhibition of 6.21 % at 4.44 mg Cs/L, i.e. an effect below 10% at the LOEC, the NOEC is not considered to be ecotoxicologically relevant. Generally, it is recommended by OECD and EFSA to apply the EC10 approach in preference to the NOEC approach for the environmental risk assessment. For further details please refer to the table given under "any other information on results including tables".
Results with reference substance (positive control):
The sensitivity of the test organism is checked twice a year in a non-GLP test using 3,5-dichlorophenol. The latest (December 2017) ErC50 value of 2.90 mg/L (nominal; 95% confidence limits: 2.66 – 3.16 mg/L) for inhibition of growth rate is in good agreement with the results of an international ring test with an ErC50 of 3.38 ± 1.30 mg/L.
Reported statistics and error estimates:
In this report numerical values are frequently rounded to a smaller degree of precision (number of digits) than used in the actual calculation. Minor differences in results obtained from calculations with such rounded values in comparison to those obtained with higher precision values are possible. They are, however, well within the limits of the experimental accuracy and thus of no practical concern. The mean values of the cell counts for each concentration plot of the exposure test were used for plotting growth curves. For the growth test, mean average growth rates were calculated (entire exposure period of 0 - 3 d). For the growth test, calculation of the percent inhibition compared to controls of growth rate or sectional growth rate [r], and yield [y] for the exposure period were performed according to the guideline. For the growth test, the percent inhibition values of growth rate and yield were plotted as a function of the test item concentration. Since the test results showed inhibition above 50 %, they were statistically analysed to determine an EC50 and EC10 values together with 95 % confidence intervals. The NOEC values were determined using appropriate statistical methods (Williams multiple sequential t-test procedure (growth rate) and multiple sequentially-rejective Welsh-t-test after Bonferroni-Holm (yield), significance level 0.05, one-sided smaller). The computer program ToxRat® was used for statistical evaluations.

Table: Inhibition of growth rate and yield compared to controls after 72 hours.

Mean measured
conc.
[mg Cs/L]

Growth rate

Yield

Growth rate [1/d]

% Inhibition

Yield [cells x 104/mL]

% Inhibition

Control

1.619

0

131.7

0

0.15

1.709

-5.60 (-)

168.5

-27.91 (-)

0.45

1.531

5.41 (-)

99.3

24.58 (-)

1.38

1.637

-1.12 (-)

135.6

-2.95 (-)

4.44

1.518

6.21 (+)

94.3

28.41 (-)

13.20

0.189

88.31 (+)

0.8

99.40 (+)

(+) significantly/ (-) not significantly different from controls (Williams multiple sequential t-test and Multiple sequentially-rejective Welsh-t-test after Bonferroni-Holm, P < 0.05, one-sided smaller)

Validity criteria fulfilled:
yes
Remarks:
Control cell number increase in 72 h was > 16 (i.e. 132.7), the mean of the control replicate CV% in the section-by-section growth rate was ≤ 35% (i.e. 2.60%) & the CV of average specific growth rate after 72 h in control replicate was ≤ 7 % (i.e. 5.73%).
Conclusions:
The potential growth(r) and yield(y)-inhibiting effect of cesium tetrafluoroaluminate on the unicellular freshwater green alga Raphidocelis subcapitata (former name Pseudokirchneriella subcapitata) was investigated in a static exposure system for 72 h according to OECD 201 (2011). Nominal concentrations of 0, 0.31, 0.93, 2.78, 8.33 and 25.0 mg test item/L have been prepared in sterile OECD medium and were chemically analysed using ICP-MS (LOQ = 0.00006 mg Cs/L; LOQ = 0.000005 mg Al/L) at the start and the end of the test. Based on cesium concentrations and following control-normalization, arithmetic mean measured concentrations of 0.15, 0.45, 1.38, 4.44 and 13.20 mg Cs/L have been calculated. Based on the cesium content of cesium tetrafluoroaluminate of 57.7 %, 72-hour ErC10 and ErC50 values of 8.53 and 14.21 mg test item/L, EyC10 and EyC50 values of 3.78 and 9.48 mg test item/L, and NOErC and NOEyC values of 2.39 and 7.69 mg test item/L were calculated, respectively.

Description of key information

In a reliable GLP- and guideline-conform study of cesium fluoroaluminate according to OECD 201 (2011), the 72-h ErC10 and ErC50 values for the inhibition of growth rate of green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) were determined with 8.53 and 14.21 mg cesium fluoroaluminate /L (based on measured Cs concentrations), respectively (Schlich, 2018). Thus, cesium fluoroaluminate seems to have some potential for toxicity to aquatic algae.

Key value for chemical safety assessment

EC50 for freshwater algae:
14.21 mg/L
EC10 or NOEC for freshwater algae:
8.53 mg/L

Additional information