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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to the analogue justification attached to IUCLID section 13
Reason / purpose:
read-across source
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
28.13 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.56 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.24 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.65 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Validity criteria fulfilled:
yes
Conclusions:
The test item caused significant inhibition of algal growth at the following concentrations:
3.2 – 100 mg/L (growth rate)
1 – 100 mg/L (yield)
NOEC for the growth rate was 1.0 mg/L and EC50 (72h) was > 28.13 mg/L based on nominal concentrations.
Executive summary:

One valid experiment was performed.

The study was performed according to OECD 201 (GLP) using 6 concentrations ranging from 0.32 to 100 mg/L. Incubation time (test system Desmodesmus subspicatus) was 72 hours.


Significant inhibition of algal growth was observed at the following concentrations:

3.2 – 100 mg/L (growth rate)

1 – 100 mg/L (yield)


At the start and at the end of the test, the content of the test item in the test solutions was determined using a photometric method. In the two lowest treatments, the values determined were below the LOQ (1 mg/L Phosphate resp.1.50 mg/L test item). The measurable concentrations lay between 95 % and 102 % of the nominal concentrations at the beginning of the test and between 59 % and 106 % of the nominal concentrations at the end of the test. These values deviate by more than ± 20% from the nominal values. Since the low values are below the LOQ, the geometric mean of the highest concentration is formed. Since the lower concentrations were prepared by dilution of the stock solution, the real concentrations were calculated on the basis of the geometric mean of the

measured concentration in the highest concentrated treatment and the respective Dilution factor. These calculated values correspond to the nominal values.

The following results for the test item were determined:

Endpoint

NOEC

LOEC

EC10

EC50

Growth Rate

1 mg/L

3.2 mg/L

0.56 mg/L

28.13 mg/L

Yield

0.32 mg/L

1 mg/L

0.65 mg/L

2.24 mg/L

Note: According to the guideline, NOEC is determined by comparing of the respective treatment with the blank control. Statistically insignificant variation is considered as “no observed effect”, although the EC10 which is read from the graph toxicity vs. concentration may lie lower.


Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06. Aug. 2018 to 09. Aug. 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
OECD Guideline for the Testing of Chemicals No. 201, adopted 23. Mar. 2006, Annex
5 corrected: 28 July 2011 “Freshwater Alga and Cyanobacteria, Growth Inhibition
Test”
Deviations:
yes
Remarks:
See section "Any other information on material and methods incl. tables" - Stock solution autoclaved for 20 min instead of 15 min. - Temperature in the Experiment - Temperature during pre-culture
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2009
Deviations:
yes
Remarks:
See section "Any other information on material and methods incl. tables"
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- CAS No.: 14306-25-3
- EC No.: 238-242-6
- Source and lot/batch No.of test material: NA2253
- Expiration date of the lot/batch: June 2019
- Purity test date: 16 May 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored in a closed vessel at room temperature (20 ± 5°C) protected from light and humidity.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.32 / 1 / 3.2 / 10 / 32 / 100 mg/L nominal concentration.The concentrations to be tested are based on non GLP pre-tests.
- Sampling method: At test start (0 h) and test end (72 h), all samples were analysed. All samples will be measured with and without disgestion.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution containing 100.2 mg/L in algal medium (demineralised water enriched with minerals but without algae) was prepared. The lower treatments were prepared by dilution
of this stock solution with algal medium.
- Controls: yes, algal medium and algae
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular freshwater green alga
- Strain: 86.81 SAG
- Source: The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM S
ciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen).
- Method of cultivation: The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
72 ± 3 hours
Hardness:
n.a.
Test temperature:
16.0 - 28.2°C
pH:
for Information see section "Any other information on materials and methods incl. tables".

In the test solutions and the blank control solution, the pH-value is measured at the start and the end of the test. The pH of the blank control should not vary by more than 1.5 Units.
Dissolved oxygen:
n.a.
Salinity:
n.a.
Conductivity:
for Information see section "Any other information on materials and methods incl. tables".
Nominal and measured concentrations:
- control / 0.32 / 1 / 3.2 / 10 / 32 / 100 mg/L test item (nominal);
- control / 0.3 / 1 / 3.2 / 10 / 32 / 100 mg/L (calculated on the basis of the geometric mean of the
measured concentration in the highest concentrated treatment and the respective dilution
factor)
for more information see section: "Any other information on materials and methods incl. tables".
Details on test conditions:
TEST SYSTEM
- Test vessel: glass flasks (total volume 65 ml)
- Type (delete if not applicable): open (covered with perforated plastic foil acting as a stopper)
- Material, size, headspace, fill volume: 65 ml glass flasks filled with 45 ± 1 mL of the respective test solution; shaken on an orbital shaker to keep the algae in suspension.
- Initial cells density: 2.3 * 103 cells/mL
- Control end cells density: 380170 cells/mL
- 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: deionised water was used to perform the OECD media
according to OECD 201
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Photoperiod: continuous lighting
- Light intensity and quality: 5000 lux (range: 4440 to 8880 Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The real cell concentration at the beginning of the test was
measured with an electronic particle counter in the blank control solution. This measured value was used as start cell concentration for all treatments. Every 24 ± 1 hours, the cell number in all vessels was measured with an electronic particle counter.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: non-GLP range-finder was performed without any additional information
- Test concentrations: 0.32 / 1 / 3.2 / 10 / 32 / 100 mg/L (nominal)
Reference substance (positive control):
yes
Remarks:
yes potassium dichromate (K2Cr2O7, CAS No. 7778-50-9)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
28.13 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.56 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.24 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.65 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The microscopic examination after 72 h of test duration did reveal a normal and healthy appearance of the algae in the test item concentrations 0.32, 1, 3.2, 10 mg/L and in the control. In the highest test item concentrations 32 and 100 mg/L no cells were visible after 72 h.
- Any stimulation of growth found in any treatment: slight groth inhibition at the lowest test item concentration 0.32 mg/L: 0.7% (growth rate) and 3.3% (yield) stimulation compared to blank control.
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate
reference test.
72h EC50: 0.79 mg/L (growth rate)
72h EC50: 0.39 mg/L (yield)
The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).
Reported statistics and error estimates:
EC values were calculated by a 3-parametric concentration distribution function. The NOEC andLOEC were determined by Dunnett’s Multiple t-test procedure. Prior to the Dunnett’s test, a Shapiro-
Wilk’s test and a Levene’s test were conducted to test for normality and homogeneity of variance,
respectively, over treatments at each time point. Calculation of results was performed with the help of validated software (Microsoft Excel®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.2.1, ToxRat Solutions GmbH.

Growth Rate, Yield

From the cell numbers, the Growth Rate μ and the Yield were calculated. The means and standard deviations at the end of the test are given in the following table:

Table Growth Rate μ, Yield

Nominal Concentration in mg/L

Parameter

Growth Rate

(0-72h) [day-1]

Yield (0-72h)
[Cell Concentration/mL]

Blank control

Mean

1.70

377870

SD

0.04

44906

0.32

Mean

1.71

390320

SD

0.02

20116

1

Mean

1.63

307047

SD

0.02

18042

3.2

Mean

1.35

131560

SD

0.09

34758

10

Mean

0.90

31633

SD

0.04

4020

32

Mean

0.77

21100

SD

0.04

2445

100

Mean

0.77

20733

SD

0.05

3251


Inhibition

The following mean inhibition values were calculated for the Growth Rate μ and the Yield.

Table Inhibition Values

Nominal concentration in mg/L

% Inhibition

Growth Rate (0-72h)

Yield (0-72h)

Blank control

0

0

0.32

-0.7

-3.3

1

3.9

18.7

3.2

20.8

65.2

10

47.3

91.6

32

54.6

94.4

100

55.0

94.5


Table Validity criteria for OECD 201.

Parameter

Validity criteria

Observed value

Assessment

Increase factor biomass

factor 16 in 72 h

165

Valid

Mean coefficient of variation of daily growth rates

max. 35%

11%

Valid

Coefficient of variation of average growth rate during the whole test period

max. 7%

2%

Valid


Biological Results

The following values were determined:

Table Biological Results of the Test Item

Parameter

Value

95% confidence interval

NOEC (Growth Rate) 72 h

1 mg/L

--

NOEC (Yield) 72 h

0.32 mg/L

--

LOEC (Growth Rate) 72 h

3.2 mg/L

--

LOEC (Yield) 72 h

1 mg/L

--

72h ErC10

0.56 mg/L

0.19 – 1.66 mg/L

72h EyC10

0.65 mg/L

0.41 – 1.04 mg/L

72h ErC50

28.13 mg/L

7.13 – 105.55 mg/L

72h EyC50

2.24 mg/L

1.31 – 3.81 mg/L


Validity criteria fulfilled:
yes
Remarks:
For further details please see section “Any other information on results incl. tables”.
Conclusions:
The test item caused significant inhibition of algal growth at the following concentrations:
3.2 – 100 mg/L (growth rate)
1 – 100 mg/L (yield)
NOEC for the growth rate was 1.0 mg/L and EC50 (72h) was > 28.13 mg/L based on nominal concentrations.
Executive summary:

One valid experiment was performed.

The study was performed according to OECD 201 (GLP) using 6 concentrations ranging from 0.32 to 100 mg/L. Incubation time (test system Desmodesmus subspicatus) was 72 hours.


Significant inhibition of algal growth was observed at the following concentrations:

3.2 – 100 mg/L (growth rate)

1 – 100 mg/L (yield)


At the start and at the end of the test, the content of the test item in the test solutions was determined using a photometric method. In the two lowest treatments, the values determined were below the LOQ (1 mg/L Phosphate resp.1.50 mg/L test item). The measurable concentrations lay between 95 % and 102 % of the nominal concentrations at the beginning of the test and between 59 % and 106 % of the nominal concentrations at the end of the test. These values deviate by more than ± 20% from the nominal values. Since the low values are below the LOQ, the geometric mean of the highest concentration is formed. Since the lower concentrations were prepared by dilution of the stock solution, the real concentrations were calculated on the basis of the geometric mean of the

measured concentration in the highest concentrated treatment and the respective Dilution factor. These calculated values correspond to the nominal values.

The following results for the test item were determined:

Endpoint

NOEC

LOEC

EC10

EC50

Growth Rate

1 mg/L

3.2 mg/L

0.56 mg/L

28.13 mg/L

Yield

0.32 mg/L

1 mg/L

0.65 mg/L

2.24 mg/L

Note: According to the guideline, NOEC is determined by comparing of the respective treatment with the blank control. Statistically insignificant variation is considered as “no observed effect”, although the EC10 which is read from the graph toxicity vs. concentration may lie lower.


Description of key information

Growth rate:

NOEC (72h): 1 mg/L (D. subspicatus,OECD 201, nominal, read-across)

EC50 (72h): 28.13 mg/L (D. subspicatus,OECD 201, nominal, read-across)

Yield:

NOEC (72h): 0.32 mg/L (D. subspicatus,OECD 201, nominal, read-across)

EC50 (72h): 2.24 mg/L (D. subspicatus,OECD 201, nominal, read-across)

Key value for chemical safety assessment

Additional information

No data are available on the toxicity towards aquatic algae and cyanobacteria of "Esters of phosphoric acid with myo-Inositol, plant-derived" (CAS No. 83-86-3).

Therefore, data from the analogue substance "Esters of sodium hydrogen phosphate with myo-Inositol, plant-derived" (CAS 14306 -25 -3; EC 238 -242 -6) were used for read across. This read-across is in accordance with Regulation (EC) No. 1907/2006 Annex XI, 1.5. In this case of read-across, the best suited (highest degree of structural similarity, nearest physico-chemical properties) read-across substance was used for the assessment. This read-across is justified in within the analogue justification in IUCLID Section 13.

 

One valid experiment was performed.

The study was performed according to OECD 201 (GLP) using 6 concentrations ranging from 0.32 to 100 mg/L. Incubation time (test system Desmodesmus subspicatus) was 72 hours.

Significant inhibition of algal growth was observed at the following concentrations:

3.2 – 100 mg/L (growth rate)

1 – 100 mg/L (yield)

At the start and at the end of the test, the content of the test item in the test solutions was determined using a photometric method. In the two lowest treatments, the values determined were below the LOQ (1 mg/L Phosphate resp.1.50 mg/L test item). The measurable concentrations lay between 95 % and 102 % of the nominal concentrations at the beginning of the test and between 59 % and 106 % of the nominal concentrations at the end of the test. These values deviate by more than ± 20% from the nominal values. Since the low values are below the LOQ, the geometric mean of the highest concentration is formed. Since the lower concentrations were prepared by dilution of the stock solution, the real concentrations were calculated on the basis of the geometric mean of the measured concentration in the highest concentrated treatment and the respective Dilution factor. These calculated values correspond to the nominal values.

 

The following results for the test item were determined:

Endpoint

NOEC

LOEC

EC10

EC50

Growth Rate

1 mg/L

3.2 mg/L

0.56 mg/L

28.13 mg/L

Yield

0.32 mg/L

1 mg/L

0.65 mg/L

2.24 mg/L

Note: According to the guideline, NOEC is determined by comparing of the respective treatment with the blank control. Statistically insignificant variation is considered as “no observed effect”, although the EC10 which is read from the graph toxicity vs. concentration may lie lower.