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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:
June 2016 -
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL:
- Chemical name: Phosphonium, tetrakis (hydroxymethyl)-,sulphate(2:1); polymer with urea
- CAS number: 63502-25-0
- Batch no.: PLFS17D1
- Expiration date of the lot/batch: 31. August 2016
- Purity: Active content (Iodine titration): 67.8% w/w / THPS (CAS number 55566-30-8): 25.7% w/w
- Solubility in water: Miscible
- pH: 5.0 at 20 °C
- Specific gravity (s.g.): 1.377 g/ml
- Appearance: Clear, yellow liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL:
- Stability under correct storage conditions (expiry date): 31. August 2016
- Storage requirements: Keep container tightly closed in a cool, dry place.
Analytical monitoring:
yes
Details on sampling:
- Sampling method:
Samples of stock and/or test solutions were taken at the start and at the end of exposure (i.e. T0 hours and T48 hours) to determine the actual test item concentrations. Control and test solutions will be sampled in duplicate. The duplicate samples will be kept separately as a reserve.

- Sample storage conditions before analysis:
After sampling and before shipment, the samples will be stored in glass bottles in the dark at a temperature of ≤ -18°C if not advised otherwise.
Samples were transferred to the test site for chemical analysis under the required storage conditions.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
At the day of the test start a stock solution (S1) was prepared by diluting 0.05027 g of the test item in 1000 mL of test medium, resulting in a nominal concentration of 50.0 mg test item/L. The stock solution (S1) was stirred for 45 minutes using a magnetic stirrer at ambient temperature, and ultras onicated for 3 min. The test solutions were then prepared by diluting the stock solution S1 with test medium.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Raphidocelis subcapitata (former name: Pseudokirchneriella subcapitata)
- Strain: SAG 61.81
- Source (laboratory, culture collection): originally supplied by Sammlung von Algenkulturen, Albrecht-von-Haller-Institut, Universität Göttingen, Germany in February 2016
- Age of inoculum (at test initiation):
- Method of cultivation:

ACCLIMATION
- Acclimation period: To adapt the algae to the test conditions a pre-culture was inoculated by a liquid algal culture and incubated under test conditions.
- Culturing media and conditions (same as test:
pH-value of the algal medium: 8.3
Culture/test/growth medium: Mod. OECD medium (OECD 201, EN ISO 8692)
Amount of liquid stock culture per pre-culture vessel: 100±5 mL
Pre-culture vessels: 300 mL Erlenmeyer flasks
Number of replicates: 2
Light: Permanent illumination (24/0 h light/dark); fluorescent tubes of universal white type
Light intensity: 60–120 µE m–2 s–1
Shaker: 100±5 oscillations/min
Temperature in the test room:
21–24 °C, controlled at ±2 °C (nominal)

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
none
Test temperature:
range from 21.8 to 22.5 during the test
pH:
range from 7.9 to 9.4 during the test for the control
Salinity:
no data
Conductivity:
no data
Nominal and measured concentrations:
Based on the results of preliminary non-GLP range-finding test, the definitive test was performed using nominal concentrations of:
- 0.0500, 0.158, 0.500, 1.58 and 5.00 mg test item/L.

These concentrations correspond to the following concentrations of active ingredient, using the content of active ingredient of 67.8% (w/w) as reported in the Certificate of Analysis:
- 0.0339, 0.107, 0.339, 1.07 and 3.39 mg active ingredient/L.

In order to calculate the highest active ingredient concentration, the highest test item concentration was multiplied by 0.678. The lower concentrations are derived by applying the respective spacing factor.
Details on test conditions:
TEST SYSTEM
- Test vessel: 300-mL Erlenmeyer flasks
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume:
- Aeration:
- Renewal rate of test solution (frequency/flow rate):
- Number of cells per mL in the pre-culture before inoculating the test solution: 63×104
- Number of cells per mL test solution at the beginning of the test: 0.5×104
- Control end cells density: At start of the test, the cell numbers were determined microscopically using a counting chamber (Thoma chamber).
After 24, 48 and 72 hours, the cell numbers were determined by measuring the fluorescence intensity in 4 samples of 200 µL of test solution per replicate using a fluorometer (Multiple Reader Tecan ULTRA).
- No. of vessels per concentration: 3 replicates
- No. of vessels per control : 6 replicates
- No. of vessels for stability check (limit concentration without algae): 2 replicates

GROWTH MEDIUM
- Standard medium used: The test medium (growth mediumn culture medium) was OECD medium as described in the test guideline, modified as the OECD growth medium was prepared with a 10-fold concentrated iron (Fe)-EDTA complex (see bold font in recipe below) compared to the original recipe according to guideline OECD 201
Before use, the pH of the medium was adjusted to 8.1±0.2. The algal medium to be used as test medium was sterilised (filtered, pore size 0.2 µm) before use. The vessels used were sterilised using a drying oven for three hours at 150 °C.

TEST MEDIUM / WATER PARAMETERS
- Water Quality Measurements:
After temperature adaptation of the test solutions, the pH was measured and recorded for each test concentration and the control in one replicate. At the end of the test, the pH was measured and recorded in pooled replicates of each concentration and the control.
Temperature was recorded automatically once per hour throughout the test in a separate test vessel placed on the shaker containing approx. 100 mL deionised water

OTHER TEST CONDITION
- Photoperiod: 24 h light/0 h dark
- Light intensity and quality: Permanent illumination / 60–120 µE m-2s-1, within ±15% over the incubation area

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations:
At start of the test, the cell numbers were determined microscopically using a counting chamber (Thoma chamber).
fter 24, 48 and 72 hours, the cell numbers were determined by measuring the fluorescence intensity in 4 samples of 200 µL of test solution per replicate using a fluorometer (Multiple Reader Tecan ULTRA).

Yield growth rate were calculated according to OECD guideline 201.

RANGE FINDING TEST
Reference substance (positive control):
yes
Remarks:
potassium dichromate (K2Cr2O7)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.151 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.275 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.256 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.037 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.698 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.406 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.378 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.054 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.039 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.05 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
A clear concentration-response relationship was observed for both biological parameters growth rate and yield during the exposure period.

Growth rate:
The ErC50 of Raphidocelis subcapitata was determined to be 1.698 mg test item/L based on nominal concentration.
The ErC50 for the active ingredient was calculated by multiplying the ErC50 for test item by 0.678, resulting in 1.151 mg a.i./L.
The NOEC based on nominal concentration was determined to be 0.050 mg test item/L, and 0.0339 mg a.i./L

Yield:
The EyC50 of Raphidocelis subcapitata was determined to be 0.378 mg test item/L based on nominal concentration.
The EyC50 for the active ingredient was calculated by multiplying the EyC50 for test item by 0.678, resulting in 0.256 mg a.i./L.
The NOEC based on nominal concentration was determined to be 0.050 mg test item/L and 0.0339 mg a.i./L


Morphological observations:
In the control (C0) and in the lowest concentration level C1 (0.050 mg test item/L) no deformed and/or damaged algal cells were observed during microscopic inspection at the end of the test. At C2 and C3 (0.158 and 0.500 mg test item/L) many deformed or enlarged cells were observed at test end. At the concentration level C4 (1.58 mg test item/L) and C5 (5.00 mg test item/L) approximately 90% of the algae cells appeared damaged and/or deformed.
Results with reference substance (positive control):
Growth rate ErC50 (0–72 h): 0.913 (0.886 – 0.940); 95%-CL) mg/L
This result is in accordance with the range given in the international ring test (ISO (2004); Water Quality – Freshwater algal growth inhibition test with unicellular green algae. European Standard EN ISO 8692, October 2004.) mentioned in OECD guideline 201, the ErC50 (72h)-values for potassium dichromate obtained from different laboratories should be 1.19 mg/L with a standard deviation of 0.27 mg/L.
Reported statistics and error estimates:
The data were evaluated by the Range-to-Standard-deviation ratio (R/S) Test for normal distribution, and by Levene's Test for homogeneity of variances. The Williams Multiple Sequential t-test for homogenous variances were applied to find out whether there were significant differences between the growth of algae in the controls and the algae exposed to the test item concentrations. The significance level was 0.05.

Probit analysis using linear maximum likelihood regression was used to calculate the ECx values as based on nominal loading rates in mg test item/L.

The statistical software package ToxRat 2.10 Professional (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf) was used for these calculations.

Table 1: Inhibition of Yield: Yield per mL (divided by 104) of Pseudokirchneriella subcapitata in relation to the concentrations of the test item and % Inhibition caused by the test item after 72 hours.

Nominal concentration
[mg test item/L]

Mean [cells per mLdivided by104]

Std. Dev.

[cells]

n

%Inhibition

Significant difference from control (Williams-t test; p ≤ 0.05)

Control

134.2

21.0

6

0.0

-

0.050

139.2

16.4

3

-3.7

-

0.158

72.7

6.3

3

45.8

+

0.500

85.9

1.3

3

36.0

+

1.58

7.2

0.1

3

94.6

+

5.00

0.9

0.1

3

99.3

+

Table 2: Inhibition of Growth Rate: Growth Rate of Pseudokirchneriellasubcapitata in relation to the concentrations of the test item and %Inhibition caused by the test item after 72 hours.

Treatment
[mg test item/L]

Mean [day-1]

Std. Dev. [day-1]

n

%Inhibition

Significant difference from control (Williams-t test; p ≤ 0.05)

Control

1.862

0.0494

6

0.0

-

0.050

1.876

0.0393

3

-0.7

-

0.158

1.661

0.0282

3

10.8

+

0.500

1.717

0.0050

3

7.8

+

1.58

0.911

0.0065

3

51.1

+

5.00

0.350

0.0198

3

81.2

+

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study, the 72h-ErC50 was 1.15 mg/L and the 72h- ErC10 was 0.28 expressed as active ingredient corresponding to 1.7 mg/L and 0.41 mg/L expressed as test substance respectively.
The 72h-NOEC (growth rate and yield) was 0.039 mg/L expressed as active ingredient (corresponding to 0.050 mg/L expressed as test substance).
Executive summary:

The effect of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72 hour static test according to OECD Guideline 201 (2006), and the method C.3. of Commission Regulation (EC) No 440/2008. The study was compliant with the GLP.

Based on results of a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to the test item over a range of nominal concentrations of 0.0500, 0.158, 0.500, 1.58 and 5.00 mg test item/L corresponding to 0.0339, 0.107, 0.339, 1.07 and 3.39 mg active ingredient/L (three replicate flasks per concentration) and a control (six replicate flasks) for 72 hours under static conditions.

According to the state of art of the analytical method, only two component of the reaction mass could be monitored; the THPS and the THPO (the last one corresponding to a degradation product).

The measured THPS concentrations were found to be lower than the Limit of quantification in all the samples. Accordingly, the results could only be based on nominal concentrations of test item and active ingredient.

A clear concentration-response relationship was observed for both biological parameters growth rate and yield during the exposure period.

NOEC and ECx values for the parameters cell number and biomass expressed in yield and growth rate are based on statistical evaluation of biological results and nominal concentrations of the test item.

The 72h-ErC50 was 1.15 mg/L and the 72h- ErC10 was 0.28 mg/L expressed as active ingredient (67.8% of test item) corresponding to 1.7 mg/L and 0.41 mg/L expressed as test item respectively.

The 72h-NOEC (growth rate and yield) was 0.034 mg/L expressed as active ingredient (corresponding to 0.050 mg/L expressed as test substance).

 

The three validity criteria of the OECD guideline 201 were fulfilled, therefore this study is considered as reliable without restrictions.

Description of key information

One reliable study is available for the Reaction mass THPS-urea monomer for this endpoint.

This study was performed to assess the effect of the Reaction mass THPS-urea monomer on the green freshwater algae Pseudokirchneriella subcapitata. It was carried out according to a standard OECD 201 guideline and was GLP compliant.

 

Based on results of a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to the test item over a range of nominal concentrations of 0.0500, 0.158, 0.500, 1.58 and 5.00 mg test item/L corresponding to 0.0339, 0.107, 0.339, 1.07 and 3.39 mg active ingredient/L (three replicate flasks per concentration) and a control (six replicate flasks) for 72 hours under static conditions.

According to the state of art of the analytical method, only two component of the reaction mass could be monitored; the THPS and the THPO (the last one corresponding to a degradation product).

The measured THPS concentrations were found to be lower than the Limit of quantification in all the samples. Accordingly, the results could only be based on nominal concentrations.

A clear concentration-response relationship was observed for both biological parameters growth rate and yield during the exposure period. The NOEC and ECx values for the parameters cell number and biomass expressed in yield and growth rate are based on statistical evaluation of biological results and nominal concentrations of the test item.

The 72h-ErC50 was1.15 mg/L and the 72h- ErC10 was 0.28 expressed in active ingredient, corresponding to 1.7 mg/L and 0.41 mg/L expressed in test substance respectively.

The 72h-NOEC (growth rate and yield) was 0.039 mg/L expressed in active ingredient, corresponding to 0.050 mg/L expressed in test substance.

 

Key value for chemical safety assessment

EC50 for freshwater algae:
1.15 mg/L

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