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Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-02-17 to 2022-04-29

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Atotech Deutschland GmbH
- Lot/batch number of test material: RR1286
- Purity, including information on contaminants, isomers, etc.: 98 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep containers tightly closed in a dry, cool and well ventilated place. Recommended storage temperature: 0 - 40 °C.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stable under recommended storage conditions
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Soluble in water

Analytical monitoring:

yes

Details on sampling:

- Sampling method: After 0 h, 24 h, 48 h and 72 h exposure, the additional vessels for chemical analysis were sampled: 2 samples of 3 mL per treatment group.

- Sample storage conditions before analysis: Of each sampled treatment, one of the analytical samples from 0 h, 24 h, 48 h and 72 h was sent
to the analytical laboratory at the test site menal GmbH for chemical analysis, in a frozen state and one transfer using an insulated box with thermal packs.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution was prepared by adding 111.8 mg test item (including a factor of 1.11 to take
into account the dilution caused by addition of the algal inoculum) to 1000 mL test medium and
shaking for 30 min using an overhead shaker at 24.2 – 24.3 °C until the test item was completely
dissolved. This stock solution was used as highest test item concentration in the test. The further test item concentrations were prepared by
diluting the stock solution with test medium according to the following scheme (spacing factor of 2).

- Test concentration separation factor: 2
- Evidence of undissolved material: The test item was completely dissolved

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Strain: Strain No. 61.81 SAG
- Source (laboratory, culture collection): originates from the Culture
Collection of Algae at the University of Goettingen.
- Age of inoculum (at test initiation): The strain used for this study has been cultured in suspension culture at Hydrotox GmbH since January 2021.
- Method of cultivation: Twice a week, the stock suspension
is inoculated into fresh Holm-Hansen medium (composition: 496 mg/L NaNO3, 39 mg/L K2HPO4,
75 mg/L MgSO4×7H2O, 36 mg/L CaCl2×2H2O, 58 mg/L Na2CO3, 10 mg/L Na2EDTA×2H2O, 3 mg/L citric acid, 3 mg/L iron citrate, 0.1144 mg/L H3BO3, 0.0724 mg/L MnCl2×4H2O, 0.0088 mg/L ZnSO4×7H2O, 0.0032 mg/L CuSO4×5H2O, 0.0010 mg/L Na2MoO4×2H2O, 0.0016 mg/L CoCl2× 6H2O) under axenic conditions to keep it in exponential growth.

ACCLIMATION
Acclimation period: Before the start of the test, 2.040 mL of the algae stock suspension was diluted with 47.960 mL test medium to obtain a concentration of 5 × 104 cells/mL. This pre-culture was incubated for 4 d at 22.3 – 22.7 °C and 90.2 µE m-² s-1 ± 6.3 % continuous lighting.
- Culturing media and conditions (same as test or not): not same as test. Holm-Hansen medium was used for culturing and OECD 201 medium was used as test media
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

The temperature during the exposure was 22.2 – 23.0 °C

pH:

The pH was 7.7 – 8.0 in the control and 7.7 – 8.4 in the test item treatment

Nominal and measured concentrations:

Nominal: 6.25, 12.5, 25, 50, 100 mg/L
Measured: 6.5, 12.6, 24.3, 48.9, 98.5

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer glass flasks 100 mL, Schott, Mainz
- Type: closed
- Initial cells density: 108.105 × 105 cells/mL
- Control end cells density: The biomass (cell concentration) in the control has increased by a factor of 36.8 and therefore ≥ 16 during the test period.
- 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: The test was performed with OECD TG 201 medium and prepared according to OECD 201 (2006)
- Culture medium different from test medium: yes
- Intervals of water quality measurement: The pH was measured at the start (0 h) and at the end (72 h) of the test. The temperature was measured continuously.

OTHER TEST CONDITIONS
- Sterile test conditions: yes. components of the medium were either autoclaved or sterile filtered.
- Adjustment of pH: no
- Light intensity and quality: The lighting was continuous with an intensity of 90.2 µE m-
² s-1 ± 6.3 % (required: 60 – 120 µE m-² s-1).


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The algal biomass was monitored by measuring the chlorophyll fluorescence after 0 h, 24 h, 48 h and 72 h. To convert the measured surrogate parameter chlorophyll fluorescence into cell concentration (the measure for biomass), a correlation factor is determined twice a year within the quality check by measuring cell concentrations of different dilutions with the Coulter Counter and the corresponding fluorescence with the microplate fluorescence reader. Chlorophyll fluorescence values are correlated with measured cell concentrations. The slope of the curve gives the conversion factor. By means of the conversion factor, the surrogate parameter chlorophyll fluorescence is converted into cell concentration
- Chlorophyll measurement: see above

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Test concentrations: 0, 6.25, 12.5, 25, 50, 100 mg/L

Reference substance (positive control):

yes

Remarks:

Reference substance was tested during routine quality assurance. Potassium dichromate was tested as reference substance in February 2022 (GLP)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: Up to 40% increase in yield was observed at lowest concentration of 6.25 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: `No significant effect observed up till highest concentration of 100 mg/L

Details on results:

-The morphology of the algal cells in the test item treatments as well as in the control showed no
obvious abnormality.
- As the measured test item concentrations are within ± 20 % of the nominal concentrations, according to OECD 201 (2006), all results are given in relation to the nominal test item concentrations

Results with reference substance (positive control):

Reference substance was tested during routine quality assurance. Potassium dichromate was tested as reference substance in February 2022 (GLP) and an EC50 (72 h) of 1.03 mg/L was obtained which is similar to the interlaboratoy EC50 mean of 1.19 mg/L.

Reported statistics and error estimates:

The effect levels after 72 h exposure were calculated with the statistical software ToxRat Professional 3.3.0

Table 1: Inhibition of yield after 24, 48 and 72 h exposure

Nominal test item concentration (mg/L)	Inhibition of yield (%)
	24 h	48 h	72 h
0	-	-	-
6.25	-3.9	-39.7	-44.2
12.5	8.9	-13.9	-19.6
25	7.4	-26.0	-29.4
50	5.3	-9.7	-21.1
100	12.7	-4.6	-3.4

Table 2: Inhibition of growth rate after 24, 48 and 72 h exposure

Nominal test item concentration (mg/L)	Inhibition of growth rate (%)
	24 h	48 h	72 h
0	-	-	-
6.25	-2.6	-12.1	-9.8
12.5	4.5	-4.9	-4.8
25	3.7	-8.6	-7.0
50	2.7	-3.4	-5.1
100	7.1	-1.8	-1.0

Validity criteria fulfilled:

yes

Conclusions:

No adverse effect of 1,3-bis(3-(1H-imidazol-1-yl)propyl)urea on algae was observed up to the highest concentration of 100 mg/L

Executive summary:

In a 72 hour acute toxicity study, the cultures of Raphidocelis subcapitata were exposed to 1,3-bis(3-(1H-imidazol-1-yl)propyl)urea at nominal concentrations of 0, 6.25, 12.5, 25, 50, 100 mg/L under static conditions in accordance with the OECD 201 guideline. Nominal concentrations were verified by analytical measurements. The EC50 values based on growth rate were > 100 mg/L.  The % growth inhibition in the treated algal culture as compared to the control ranged from 1 to 9.8%.

The morphology of the algal cells in the test item treatments as well as in the control showed no obvious abnormality.

This toxicity study is classified as acceptable and satisfies the guideline requirements for OECD 201 toxicity study.

 

Results Synopsis

Test Organism: Raphidocelis subcapitata 

Test Type: static

72 hr EC50: > 100 mg/L  

 

Description of key information

The 72-h growth-rate EC50 value for Raphidocelis subcapitata was > 100 mg/L.

 

Additional information

In a 72 hour acute toxicity study, the cultures of Raphidocelis subcapitata were exposed to 1,3-bis(3-(1H-imidazol-1-yl)propyl)urea at concentrations up to 100 mg/L under static conditions in accordance with the OECD 201 guideline.  The EC50 value based on growth rate was >100 mg/L. Therefore no hazard was identified.