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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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 4133/00-K0
- Expiration date of the lot/batch: June 07, 2021
- Purity test date: Certificate of analysis dated December 03, 2020
- Content: 1.82 % Pt
- Form: liquid
- Appearance: Clear, yellowish liquid

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L, equivalent to 0.114, 0.228, 0.455, 0.910 and 1.82 mg Pt/L (spacing factor: 2).
- Sampling method:
Samples (at least 2 of about 20 mL) were taken from all test solutions and controls at the beginning of the test prior to addition of the algae.
At the end of the growth test, samples were taken directly from representative replicates per test concentration and controls.
One set of samples was filtered through a 0.2 µm PES filter before measurement. The other set of samples were measured without filtration. In addition, for each set of samples a set of retain samples was taken.
- Sample storage conditions before analysis:
All samples were stabilised by acidification and stored at 4 ± 3 °C until further analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
An amount of 100.0 mg of the test item was transferred into a sterile glass flask filled up to 1 L with growth medium (sterile) to obtain the highest test concentration.
The test media was stirred for 2 hours at room temperature (about 20°C). The highest test concentration then was serially diluted with sterile growth medium to obtain the other test concentrations. For the control growth medium without test item was used. All work was conducted on a clean bench using sterile equipment.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: unicellular green alga
- Strain: Chlorophycea, Chlorophyta
- Source (laboratory, culture collection): SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, D- 37073 Göttingen, Catalog No 61.81 SAG.
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Not stated

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

not applicable

Test temperature:

21 - 24 °C

pH:

The pH of the controls remained 8.00 ± 0.06 during the test. In the test concentrations, the initial pH in the treatments ranged from 7.18 to 7.99 and the mean pH value was between 7.76 and 7.96 at the end of the test.

Dissolved oxygen:

not stated

Salinity:

not applicable

Conductivity:

not stated

Nominal and measured concentrations:

NOMINAL: 0.114, 0.228, 0.455, 0.910 and 1.82 mg Pt/L (equivalent to 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L)

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks
- Type: open
- Material, size, headspace, fill volume: 250 mL conical glass flasks covered with air-permeable silicone-sponge caps.
- Aeration: Not stated
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): None
- Initial cells density: 1 x 10 (4) cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: not applicable

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Not stated
- Light intensity and quality: 60 - 120 µE m-2 s-1 (4440 - 8880 lux)
- Salinity (for marine algae): Not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: electronic particle counter (CASY TT, OMNI Life Science GmbH & Co. KG, Bremen, Germany).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: Yes
- Test concentrations (range-finder): 1, 10 and 100 mg/L test item
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

The sensitivity of the test organism is routinely checked using 3,5-dichlorophenol following internal SOPs in a non-GLP test twice a year. The latest (February 2021) test is in good agreement with the results of an international ring test.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

67.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other:

Remarks:

yield

Details on results:

A study was performed to determine the toxicity of Pt concentrate P on the growth of the unicellular freshwater green alga Raphidocelis subcapitata.
Exponentially-growing cultures of the green alga were exposed to five concentrations of the test item plus a control over several generations under defined conditions for 72 hours according to the OECD guideline 201.
The nominal test concentrations were prepared with sterile growth medium under sterile conditions. For the examination of the inhibition of alga growth, eight replicates of the control (test medium only) and four replicates of each treatment were tested at the following nominal concentrations: 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L, equivalent to 0.114, 0.228, 0.455, 0.910 and 1.82 mg Pt/L (spacing factor: 2).
The concentrations of the test item in the media were confirmed by ICP-MS measurements at test initiation and test termination after 72 h. The LOQ was at 0.004 µg Pt/L at test initiation and 0.021 µg Pt/L at test termination.
At test initiation, the measured concentrations ranged from 109% to 112% of nominal concentrations and therefore were within a range of 80 – 120% of the nominal concentrations. The measured concentrations remained stable within ± 20% throughout the test period of 72 hours (% of initial: 95.2 – 101%; % of nominal: 104 – 112%). Therefore, the evaluation of biological results was based on the nominal test concentrations.
A concentration-dependent inhibiting effect on the growth of the freshwater green algae was observed over the range of the tested concentrations.
The respective 72-hour ErC50 for the inhibition of growth rate was determined to be above the highest test concentration of 100 mg test item/L (extrapolated value: 103 mg test item/L). For yield, an EyC50 of 67.7 mg test item/L was determined. The EC10 values for growth rate and yield were at 62.1 and 46.3 mg test item/L, respectively.
The NOEC for growth rate was set to be at 50.0 mg test item/L (by expert judgement), whereas it was at 25.0 mg test item/L for yield.

The results are summarized in Table 1.

Results with reference substance (positive control):

Test results from February 2021: ErC50 value of 2.57 mg/L (nominal; 95% confidence limits: 2.24 – 2.95 mg/L) 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:

Since the measured concentrations were within a range of 80 – 120% of the nominal concentrations at test start and remained stable throughout the test period of 72 hours, biological evaluations were based on nominal concentrations of the test item.
Biological data were statistically analysed to determine EC50 and EC10 values together with 95 % confidence intervals, where possible. The ErC50 and EyC50 were determined using a 3-parametric non-linear regression procedure in accordance to the recommendations in the OECD 54.
The NOEC for growth rate was determined by a Williams Multiple Sequential t-test Procedure (significance level of 0.05, one-sided smaller).
The NOEC for yield was determined by the Dunnett´s multiple t-test procedure (significance level of 0.05, one-sided smaller).
The computer program ToxRat® was used for statistical evaluations.

Table 1: Effective concentrations based on nominal test concentrations for the exposure of R. subcapitata for 72 hours.

Parameter	[mg test item/L]	EC50	EC20	EC10	LOEC	NOEC
Growth rate	Value	>100^a	73.8	62.1	100^b	50.0^b
	Lower 95 %-cl	92.0	68.1	57.3
	Upper 95 %-cl	114	80.1	67.4
Yield	Value	67.7	52.7	46.3	50.0	25.0
	Lower 95 %-cl	58.4	46.7	40.8
	Upper 95 %-cl	78.3	59.5	52.5

^a         The highest inhibition on growth rate was found at 47.6% at 100 mg test item/L. The E_rC₅₀ was extrapolated to be at 103 mg test item/L and therefore was stated to be above the highest test concentration of 100 mg test item/L.

^b         The NOEC for growth rate was calculated to be at 12.5 mg test item/L. However, due to the low inhibition of 2.3% at 25.0 mg test item/L and 4.0% at 50.0 mg test item/L, the NOEC was set to be at 50.0 mg test item/L by expert judgement, since effects below 10% compared to the control are generally not considered to be ecotoxicologically relevant. The LOEC was shifted accordingly to the next higher concentration due to the change of the NOEC. It is generally recommended by OECD and EFSA to use the EC₁₀ approach in preference to the NOEC approach for the environmental risk assessment.

 

Other observations

Microscopic examinations were performed to verify a normal and healthy appearance of the inoculum culture and to detect any abnormal appearance of the algae (as may be caused by the exposure to the test substance) at the end of the test.

Microscopic observation of the algae cells at test initiation and termination of the 72 h growth test.

Date	Nominal conc. [mg test item/L]	Observations
Test start	Normal appearance of inoculum culture for all test concentrations.
Test end, 72 h	Control	Normal appearance of algal cells.
	6.25	Normal appearance of algal cells.
	12.5	Normal appearance of algal cells.
	25.0	Normal appearance of algal cells.
	50.0	Normal appearance of algal cells.
	100	Reduced number of algae cells.

Validity criteria fulfilled:

yes

Conclusions:

Based on nominal test item concentrations, the 72 hour ErC50 and the ErC10 values (growth rate) were > 100 and 62.1 mg test item/L, respectively. The 72 hour EyC50 and the EyC10 values (yield) were 67.7 and 46.3 mg test item/L, respectively. The NOEC for growth rate was set to be at 50.0 mg test item/L (by expert judgement), whereas it was at 25.0 mg test item/L for yield.

Executive summary:

A 72-h algal growth inhibition study was conducted for Pt concentrate P. The study is reliable without restrictions, being GLP-compliant and having followed standard test guidelines (OECD 201).

A concentration-dependent inhibiting effect on the growth of the freshwater green algae was observed over the range of the tested concentrations.

The respective 72-hour ErC50 for the inhibition of growth rate was determined to be above the highest test concentration of 100 mg test item/L (extrapolated value: 103 mg test item/L). For yield, an EyC50 of 67.7 mg test item/L was determined. The EC10 values for growth rate and yield were at 62.1 and 46.3 mg test item/L, respectively.

The NOEC for growth rate was set to be at 50.0 mg test item/L (by expert judgement), whereas it was at 25.0 mg test item/L for yield.

Description of key information

Based on nominal test item concentrations, the 72 hour ErC50 and the ErC10 values (growth rate) were > 100 and 62.1 mg test item/L, respectively. The 72 hour EyC50 and the EyC10 values (yield) were 67.7 and 46.3 mg test item/L, respectively. The NOEC for growth rate was set to be at 50.0 mg test item/L (by expert judgement), whereas it was at 25.0 mg test item/L for yield.

Additional information

A 72-h algal growth inhibition study was conducted for Pt concentrate P. The study is reliable without restrictions, being GLP-compliant and having followed standard test guidelines (OECD 201).

A concentration-dependent inhibiting effect on the growth of the freshwater green algae was observed over the range of the tested concentrations.

The respective 72-hour ErC50 for the inhibition of growth rate was determined to be above the highest test concentration of 100 mg test item/L (extrapolated value: 103 mg test item/L). For yield, an EyC50 of 67.7 mg test item/L was determined. The EC10 values for growth rate and yield were at 62.1 and 46.3 mg test item/L, respectively.

The NOEC for growth rate was set to be at 50.0 mg test item/L (by expert judgement), whereas it was at 25.0 mg test item/L for yield.