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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:
04 May 2018 - 15 November 2018
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
Annex 5 corrected 28 July 2011
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Test item information
Identification: Photomer 4250
Appearance: Colourless to pale yellow liquid
Batch:17C13003
Purity/Composition: UVCB
Test item storage: At room temperature protected from light
Stable under storage conditions until:14 March 2019 (expiry date)

Additional information
Test Facility test item number: 209236/A
Purity/Composition correction factor: No correction factor required
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. The method of analysis is described in a separate report
Frequency at t=0 h and t=72 h.
Volume 2.0 mL from the approximate centre of the test vessels.
Storage Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at a WAF prepared at a loading rate of 10 mg/L but without algae and samples for analysis were taken at the start and at the end of the test period.

Test Samples
The samples were analyzed on the day of sampling and were diluted in a 1:1 (v:v) ratio with acetonitrile and analyzed. When necessary, the samples were further diluted with 50/50 (v/v) acetonitrile/M2-medium to obtain concentrations within the calibration range.

Preparation of Solutions

Stock and Spiking Solutions
Stock solutions of the test item were prepared in acetonitrile at concentrations of 1000 and 2000 mg/L.
Spiking solutions were made up from a stock solution and/or dilutions of this solution. The solvent of the spiking solutions was acetonitrile.

Calibration Solutions
Six calibration solutions in the concentration range of 0.04 – 6 mg/L were prepared from two stock solutions. The end solution of the calibration solutions was 50/50 (v/v) acetonitrile/water.

Quality Control (QC) Samples
2 mL blank medium was spiked with the test item at a target concentration of 0.1, 0.5 or 100 mg/L. The QC samples were treated similarly as the test samples.
Blank QC samples consisting of blank medium were treated similarly to the QC and test samples.

Vehicle:
no
Details on test solutions:
The batch of Photomer 4250 tested was a colourless to pale yellow liquid UVCB and not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item. The preparation of test solutions was performed under dimmed light.

Preparation of test solutions started with loading rates individually prepared at 1.0 to 100 mg/L. An overnight period of magnetic stirring was applied to ensure maximum dissolution of the test item in test medium. The obtained mixtures were allowed to settle for a period of three hours. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure.

After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.
Any residual volumes were discarded.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Source: In-house laboratory culture.
Reason for selection: This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
21-23 °C
pH:
7.8-8.0
Nominal and measured concentrations:
WAFs individually prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L.
Measured concentrations were determined as 0.2, 0.87, 2.6, 10, 12 mg/L.
Details on test conditions:
Stock culture
Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

Stock culture medium M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500mg/L
K2HPO4 39.5mg/L
MgSO4.7H2O 75mg/L
Na2CO3 20mg/L
C6H8O7.H2O 6mg/L
NH4NO3 330mg/L
CaCl2.2H2O 35mg/L
C6H5FeO7.xH2O 6mg/L
H3BO3 2.9mg/L
MnCl2.4H2O 1.81mg/L
ZnCl2 0.11mg/L
CuSO4.5H2O 0.08mg/L
(NH4)6Mo7O24.4H2O 0.018mg/L

Pre-culture
3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Pre-culture medium
M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15mg/L
MgCl2.6H2O 12mg/L
CaCl2.2H2O 18mg/L
MgSO4.7H2O 15mg/L
KH2PO4 1.6mg/L
FeCl3.6H2O 64µg/L
Na2EDTA.2H2O 100µg/L
H3BO3 185µg/L
MnCl2.4H2O 415µg/L
ZnCl2 3µg/L
CoCl2.6H2O 1.5µg/L
CuCl2.2H2O 0.01µg/L
Na2MoO4.2H2O 7µg/L
NaHCO3 50mg/L
Hardness (Ca+Mg) 0.24mmol/L (24 mg CaCO3/L)
Reference substance (positive control):
yes
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
3.3 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
other: EyC50
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on statistical significance
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.87 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on biological relevance
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: based on statistical significance and biological relevance
Validity criteria fulfilled:
yes
Conclusions:
In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), Photomer 4250 inhibited the growth rate of this fresh water algae species significantly at average concentrations of 0.87 mg/L and higher.
The 72h-EL50 for growth rate inhibition (ERL50) was 12 mg/L with a 95% confidence interval ranging from 12 to 13 mg/L.
The 72h-EC50 for growth rate inhibition (ERC50) was 3.3 mg/L with a 95% confidence interval ranging from 3.1 to 3.5 mg/L.
The 72h-EC50 for yield inhibition (EYC50) was 1.0 mg/L with a 95% confidence interval ranging from 0.98 to 1.1 mg/L.
The 72h-NOEC for growth rate inhibition was 0.20 mg/L based on statistical significance.
The 72h-NOEC for growth rate inhibition was 0.87 mg/L based on biological relevance.
The 72h-NOEC for yield inhibition was below 0.20 mg/L based on statistical significance and biological relevance.
The 72h-EL50 for growth rate inhibition (ERL50) was 12 mg/L with a 95% confidence interval ranging from 12 to 13 mg/L.
Executive summary:

The objective of the study was to evaluate Photomer 4250 for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10, EC20 and EC50 for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

The batch of Photomer 4250 tested was a colourless to pale yellow liquid mixture and not completely soluble in test medium at the loading rates initially prepared. Water Accommodated Fractions (WAFs) were individually prepared at loading rates ranging between 1.0 and 100 mg/L and used as test concentrations.

A final test was performed based on the results of a preceding combined limit/range-finding test. Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to WAFs individually prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L. The initial algal cell density was 104 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start and after 72 hours of exposure.

Inhibition of growth rates and yield increased with increasing concentration of Photomer 4250 from 0.20 mg/L upwards resulting in 94 and 100% inhibition, respectively, at 12 mg/L.

Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were between 0.43 and 13 mg/L in WAFs prepared at loading rates of 1.0 to 100 mg/L, respectively. At the end of the test, the measured concentrations were at 21-92% of the initial concentrations whereby the higher concentrations tended to remain more stable than the lower test concentrations. Based on these results, the average exposure concentrations were calculated to be 0.20, 0.87, 2.6, 10 and 12 mg/L in WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L, respectively, and used to determine the effect parameters.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters obtained in this study are summarized in the table below.

Parameter (mg/L)                     NOEC*              NOEC#                     EC10              EC20              EC50

Growth rate       Value                     0.20                     0.87                     1.3                    1.8              3.3

                           lower 95%-cl                                                                1.1                    1.6              3.1

                           upper 95%-cl                                                                1.4                    1.9             3.5

Yield       Value                                   <0.20              <0.20                    0.21                  0.36           1.0

                           lower 95%-cl                                                                0.18                  0.32           0.98

                           upper 95%-cl                                                                0.23                  0.40            1.1

cl – confidence limit, * based on statistical significance, # based on biological relevance.

In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), Photomer 4250 inhibited the growth rate of this fresh water algae species significantly at average concentrations of 0.87 mg/L and higher. The 72h-EC50 for growth rate inhibition (ERC50) was 3.3 mg/L with a 95% confidence interval ranging from 3.1 to 3.5 mg/L. The 72h-EC50 for yield inhibition (EYC50) was 1.0 mg/L with a 95% confidence interval ranging from 0.98 to 1.1 mg/L. The 72h-EL50 for growth rate inhibition (ERL50) was 12 mg/L with a 95% confidence interval ranging from 12 to 13 mg/L.

The effect loading rate (EL50) for growth rate inhibition has to be considered when determining the classification under (EC) No. 1272/2008 due to Photomer 4250 being a UVCB and not completely soluble in test medium at the loading rates initially prepared. Water Accommodated Fractions (WAFs) were individually prepared at loading rates ranging between 1.0 and 100 mg/L and used as test concentrations. The 72h-EL50 for growth rate inhibition (ERL50) was 12 mg/L with a 95% confidence interval ranging from 12 to 13 mg/L. Based on this result Photomer 4250 is classified as aquatic toxicity chronic category 3.

Description of key information

The batch of Photomer 4250 tested was a colourless to pale yellow liquid UVCB and not completely soluble in test medium at the loading rates initially prepared. Water

Accommodated Fractions (WAFs) were individually prepared at loading rates ranging between 1.0 and 100 mg/L and used as test concentrations. Therefore, the effect loading rate (EL50) for growth rate inhibition has to be considered when determining the classification under (EC) No. 1272/2008.

The 72h-EL50 for growth rate inhibition (ERL50) was 12 mg/L with a 95% confidence interval ranging from 12 to 13 mg/L. Based on this result Photomer 4250 is classified as aquatic toxicity chronic category 3

Key value for chemical safety assessment

Additional information

The 72h-EL50 for growth rate inhibition (ERL50) was 12 mg/L with a 95% confidence interval ranging from 12 to 13 mg/L.