Orthoperiodic acid

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

28 September 2012 to 09 December 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A read-across justification report (RAAF) will be added to Section 13 as soon as possible.

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples were taken from the control (replicates R1 - R6 pooled) and each test group (replicates R1 - R3 pooled).
- Sampling method: Samples were taken at 0 and 72 hours for quantitative analysis.
- Sample storage conditions before analysis: All samples were stored at approximately -20 °C prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately -20 °C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
50 mg of test material was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made. An aliquot of each of the stock solutions was separately inoculated with algal suspension to give the required test concentrations.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10³ cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 - 10^5 cells/mL.

ACCLIMATION
- Culturing media and conditions: The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

7.4 - 8.2

Nominal and measured concentrations:

0.10, 0.32, 1.0, 3.2, 10 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type: closed (fasks were plugged with polyurethane foam bungs)
- Fill volume: 100 mL
- Aeration: flasks were constantly shaken at aproximately 150 rpm
- Initial cells density: 5 x 10³ cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes. NaNO3 25.5 mg/L, MgCl2.2H2O 12.164 mg/L, CaCl2.2H2O 4.41 mg/L, MgSO4.7H2O 14.7 mg/L, K2HPO4 1.044 mg/L, NaHCO3 15.0 mg/L, H3BO3 0.1855 mg/L, MnCl2.4H2O 0.415 mg/L, ZnCL2 0.00327 mg/L, FeCl3.6H2O 0.159 mg/L, CoCl2.6H2O 0.00143 mg/L, Na2MoO4.2H2O 0.00726 mg/L, CuCl2.2H2O 0.000012 mg/L and Na2EDTA 0.30 mg/L prepared in reverse osmosis purified water

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified water
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuously illuminated
- Light intensity and quality: approximately 7000 lux (provided by warm white lighting (380 - 730 nm)

EFFECT PARAMETERS MEASURED
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

TEST CONCENTRATIONS
- Range finding study
The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test material was dissolved directly in culture medium. The control group was maintained under identical conditions but not exposed to the test material. At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated at 24 ± 1 ºC under continuous illumination and constantly shaken at approximately 150 rpm for 72 hours. After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
- Test concentrations: 0.10, 1.0, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at the test concentrations of 0.10 mg/L. However, growth was observed to be reduced at 1.0, 10 and 100 mg/L.
Based on this information test concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L were selected for the definitive test.

EVALUATION OF DATA
- Comparison of Growth Rates
The average specific growth rate for a specified period was calculated as the logarithmic increase in biomass from the equation:

µ = (ln Nn - ln N1) / (tn - t1)

where
μ = average specific growth rate from time t1 to tn (cells/mL/hour)
N1 = cell concentration at t1 (cells/mL)
Nn = cell concentration at tn (cells/mL)
t1 = time of first measurement (hours)
tn = time of nth measurement (hours)

The average specific growth rate over the test duration was calculated for each replicate control and test material vessel using the nominally inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.

In addition the section by section specific growth rate (days 0-1, 1-2 and 2-3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant.

Percentage inhibition of growth rate for each replicate test item vessel was calculated using the following equation:

Ir = [(µc - µt) / µc] x 100

where
Ir = percentage inhibition of average specific growth rate
μc = mean average specific growth rate for the control cultures (cells/mL/hour)
μt = average specific growth rate for the test culture (cells/mL/hour)

- Comparison of Yield
Yield was calculated as the increase in biomass over the exposure period using the following equation:

Y = Nn - N0

where
Y = yield (cells/mL)
N0 = cell concentration at the start of the test (cells/mL at t0)
Nn = cell concentration at the end of the test (cells/mL at tn)

For each test concentration and control the mean value for yield along with the standard deviation was calculated. Percentage inhibition of yield was calculated using the following equation:

Iy = [(Yc - Yt) / Yc] x 100

where
Iy = percentage inhibition of yield
Yc = mean value for yield in the control group (cells/mL)
Yt = mean value for yield for the treatment group (cells/mL)

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.85 - 1.5 mg/L (95 % CL)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The growth rate (r) and yield (y) of Pseudokirchneriella subcapitata were affected by the presence of the test material over the 72-hour exposure period.

- Growth data
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control. There were no statistically significant differences between the control and 0.10 mg/L test concentration (P ≥ 0.05), however, all other test concentrations were significantly different (P < 0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.10 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 0.32 mg/L.

- Yield data
There were no statistically significant differences between the control and 0.10 mg/L test concentration (P ≥ 0.05), however, all other test concentrations were significantly different (P < 0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.10 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 0.32 mg/L.

- Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.10 and 0.32 mg/L, however cell debris was observed to be present in the test cultures at 1.0, 3.2 and 10 mg/L.

- Observations on Test Material Solubility
At the start of the test all control cultures were observed to be clear colourless solutions. The test cultures were observed to range from very pale yellow solutions at 0.10 mg/L through to pale yellow solutions at 10 mg/L. After the 72-hour test period all control, 0.10 and 0.32 mg/L test cultures were observed to be pale green dispersions whilst the 1.0, 3.2 and 10 mg/L test cultures were observed to be very pale yellow solutions.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72 hour EC50 for the growth rate = 1.1 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package.

Table 1: Cell Densities and Percentage Inhibition of Growth from the Initial Range-Finding Test

Nominal conc. (% v/v saturated solution)	Cell densities* (cells/mL)		Inhibition values (%)
	0 hours	72 hours	Growth rate	Yield
Control	4.57E+03	5.95E+05	-	-
0.10	4.02E+03	5.39E+05	0	9
1.0	4.25E+03	7.98E+04	40	87
10	4.12E+03	8.55E+03	85	99
100	3.62E+03	3.36E+03	101	100

*Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

Table 2: Cell Densities and Percentage Inhibition of Growth from the Definitive Test

Nominal conc. (% v/v saturated solution)	Cell densities* (cells/mL)				Growth rate
	0 hours	24 hours	48 hours	72 hours	0- 72 hours	% inhibition
Control	5.17E+03	2.62E+04	1.55 E+05	8.20 E+05	0.071	-
0.10	5.11 E+03	3.00 E+04	1.79 E+05	7.69 E+05	0.070	1
0.32	5.04 E+03	2.79 E+04	1.35 E+05	5.62 E+05	0.066	8
1.0	5.11 E+03	1.46 E+04	3.13 E+04	6.88 E+04	0.036	49
3.2	5.03 E+03	6.42 E+03	1.50 E+04	1.65 E+04	0.016	77
10	5.39 E+03	3.40 E+03	4.66 E+03	2.67 E+03	-0.009	113

*Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 72 hour EC50, based on growth rate, was determined to be 1.1 mg/L. The No Observed Effect Concentration, based on growth rate, was determined to be 0.10 mg/L.

Executive summary:

The toxicity of the test material to aquatic algae was investigated in a GLP study which was conducted in accordance with standardised guidelines OECD 201 and EU Method C.3.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 89 to 96 % of nominal and so the results are based on nominal test concentrations only.

Under the conditions of the study, the 72 hour EC50, based on growth rate, was determined to be 1.1 mg/L. The No Observed Effect Concentration, based on growth rate, was determined to be 0.10 mg/L.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

A read-across justification report (RAAF) will be added to Section 13 as soon as possible.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.85-1.5 mg/L (95 % CL)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 October 2018 to 21 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

28 July 2011

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency: At t=0 h, t=24 h and t=72 h.
Volume: 4.8 mL from the approximate centre of the test vessels.
Storage: Samples were stored in a freezer (≤ -15 °C) until analysis at the analytical laboratory of the Test Facility.
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 1.0 mg/L but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Additionally, reserve samples of 4.8 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤ -15 °C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The batch of the test material tested was completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test material.
Preparation of test solutions started with the highest concentration of 100 mg/L. No other treatment than vigorous shaking was needed to completely dissolve the test material in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All lower test solutions were clear and colorless at the end of the preparation procedure. Test solutions of 1.0 mg/L and higher were clear and very slightly yellow to light yellow.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata)
- Strain: NIVA CHL 1
- Source: In-house laboratory culture.
- Method of cultivation: 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. The light intensity was 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
- 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.

ACCLIMATION
- Culturing media and conditions: The pre-culture was maintained under the same conditions as used in the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 - 24 °C ± 1 °C

pH:

7.5 - 8.4

Nominal and measured concentrations:

0.10, 0.32, 1.0, 3.2 and 10 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Type: Aluminium caps were perforated for ventilation
- Material, size, headspace, fill volume: Glass, 50 mL test solution
- Initial cells density: 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.
- No. of vessels per concentration: 3 replicates of each test concentration.
- No. of vessels per control: 6 replicates of the control.
- No. of vessels per vehicle control: 1 or 2 replicates of each test concentration without algae.
- 1 extra replicate of each test group for sampling purposes after 24 hours of exposure

GROWTH MEDIUM
- Detailed composition of 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:
500 mg/L NaNO3, 39.5 mg/L K2HPO4, 75 mg/L MgSO4.7H2O, 20 mg/L Na2CO3; 6 mg/L C6H8O7.H2O, 330 mg/L NH4NO3, 35 mg/L CaCl2.2H2O, 6 mg/L C6H5FeO7.xH2O, 2.9 mg/L H3BO3, 1.81 mg/L MnCl2.4H2O, 0.11 mg/L ZnCl2, 0.08 mg/L CuSO4.5H2O, 0.018 mg/L (NH4)6Mo7O24.4H2O.
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 10^4 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:
15 mg/L NH4Cl, 12 mg/L MgCl2.6H2O, 18 mg/L CaCl2.2H2O, 15 mg/L MgSO4.7H2O, 1.6 mg/L KH2PO4, 64 µg/L FeCl3.6H2O, 100 µg/L Na2EDTA.2H2O, 185 µg/L H3BO3, 415 µg/L MnCl2.4H2O, 3 µg/L ZnCl2, 1.5 µg/L CoCl2.6H2O, 0.01 µg/L CuCl2.2H2O, 7 µg/L Na2MoO4.2H2O, 50 mg/L NaHCO3. Hardness (Ca+Mg): 0.24 mmol/L (24 mg CaCO3/L); pH: 8.1 ± 0.2.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA).

OTHER TEST CONDITIONS
- pH: Measured at the beginning and at the end of the test, for all test concentrations and the control.
- Temperature of medium: Measured continuously in a temperature control vessel.
- Photoperiod: Continuous
- Light intensity and quality: TLD-lamps with a light intensity within the range of 86 to 90 µE.m^-2.s^-1.
- Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
- Appearance of the cells: At the end of the final test microscopic observations were performed on the 1.0 mg/L test concentration and the control to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Range finding study: The project started with a combined limit/range-finding test. Six replicates of exponentially growing algae were exposed to a control and a concentration of 100 mg/L. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Three replicates per concentration were exposed to 0.10, 1.0 and 10 mg/L in the combined range-finding test.
• Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.
• One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval.
• At the end of the test algae were not observed under a microscope to verify a normal and healthy appearance.
- Test concentrations: 0.10, 1.0 and 10 mg/L in the combined range-finding test.
- Results used to determine the conditions for the definitive study: No inhibition of algal growth rate or yield was found at the lowest test material concentration. Dose-related inhibition of algal growth rate and yield was found at 1.0 mg/L upwards at the end of the test. An inhibition of 87 and 99 % was observed at the highest test concentration, respectively.
Based on these results, samples taken from nominally 0.10 and 100 mg/L were analysed. The measured concentrations at the start of the test were 0.10 and 110 mg/L, respectively. During the exposure period, the 100 mg/L concentration remained stable, i.e. was at 101 % relative to initial at the end of the test. The 0.10 mg/L concentration decreased to 38 % of initial at the end of the test.
All test conditions were maintained within the limits prescribed by the study plan.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence interval from 2.4 to 2.7 mg/L.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Observation of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 1.0 mg/L when compared to the control.
- Measured test material concentrations: Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 0.096, 0.32, 1.0, 3.4 and 11 mg/L, respectively. During the exposure period, the concentrations of 1.0 mg/L and higher remained stable, i.e. were at 93 - 104 % relative to initial at the end of the test. The two lowest test concentrations (i.e. nominal concentrations of 0.10 and 0.32 mg/L, respectively) decreased to 32 and 68 % of initial at the end of the test.
It should be noted that small responses were detected in the control throughout the test. These responses were probably caused by carry over. Considering that the concentration could be estimated only by extrapolation of the calibration curve and was 1.2 – 4.6 % of the concentration measured at the lowest test concentration, this was considered negligible.
Based on these results, the average exposure concentrations were calculated and used to express effect parameters.
The concentrations measured in the samples taken from solutions with algae were comparable with the concentrations measured in the samples without algae. Hence, it can be stated that the presence of the algae did not affected the concentration of the test item in test medium throughout the test.

- Inhibition of Growth Rate and Inhibition of Yield: Inhibition of growth rates and yield increased with increasing concentration of the test material resulting in 78 % growth rate inhibition and 99 % yield inhibition at 11 mg/L. Statistically significant inhibition of growth rates was found at test concentrations of 1.0 mg/L and higher. Statistically significant inhibition of yield was found at all test concentrations.

Results with reference substance (positive control):

The objective of the study was to evaluate Potassium dichromate for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) (strain: NIVA CHL-1) during an exposure period of 72 hours and, if possible, to determine the EC50 for inhibition of both growth rate and yield (Charles River Den Bosch Study Number 20174201).
Algae were exposed for a period of 72 hours to K2Cr2O7 (Potassium dichromate) concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L and to a control. The initial cell density was 1.0 x 10^4 cells/mL.
The study met the acceptability criteria prescribed by the study plan and was considered valid.

Potassium dichromate inhibited growth rate of this fresh water algae species at all concentrations tested.
The EC50 for growth rate inhibition (72 h-ErC50) was 1.05 mg/L with a 95 % confidence interval ranging from 1.04 to 1.06 mg/L.
The historical ranges for growth rate inhibition lie between 0.82 and 2.6 mg/L. Hence, the 72 h-ErC50 for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (72 h-EyC50) was 0.295 mg/L with a 95 % confidence interval ranging from 0.292 to 0.298 mg/L.

Reported statistics and error estimates:

Determination of the NOEC and Calculation of the EC50: For determination of the NOEC and the ECx the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller) or inhibition of yield (Step-down Jonckheere-Terpstra Test Procedure, α=0.05, one-sided, smaller).
Calculation of ECx-values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test material.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Time Weighted Average Versus Nominal Concentrations

Test Material Nominal Conc. (mg/L)	Measured concentration (mg/L)			TWA (mg/L)
	t=0h	t=24h	t=72 h
0.10	0.096	0.098	0.031	0.069
0.32	0.32	0.33	0.22	0.29
1.0	1.0	1.0	0.96	1.0
1.0WA	1.1	1.0	1.1	1.1
3.2	3.4	3.5	3.5	3.5
10	11	11	11	11

WA – Without Algae.

 

Growth Rate and Percentage Inhibition for the Total Test Period

Test Material TWA Conc. (mg/L)	Mean	Std. Dev.	n	% Inhibition
Control	1.749	0.0139	6
0.069	1.702	0.0178	3	2.7
0.29	1.710	0.0504	3	2.2
1.0	1.522	0.0297	3	13*
3.5	0.481	0.0330	3	73*
11	0.393	0.0595	3	78*

* - Effect was statistically significant.

 

Growth Rate and Percentage Inhibition at Different Time Intervals

Test Material TWA Conc. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72 h
		Mean	% Inhibition	Mean	% Inhibition	Mean	% Inhibition
Control	6	1.911		1.726		1.610
0.069	3	1.911	0.0	1.659	3.8	1.536	4.6
0.29	3	1.722	9.9	1.900	-10	1.510	6.2
1.0	3	1.497	22	1.410	18	1.661	-3.2
3.5	3	1.409	26	0.236	86	-0.201	113
11	3	1.081	43	0.162	91	-0.063	104

 

Yield and Percentage Inhibition for the Total Test Period

Test Material TWA Conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	189.0	7.93	6
0.069	164.3	8.73	3	13*
0.29	169.5	24.66	3	10*
1.0	95.6	8.52	3	49*
3.5	3.2	0.41	3	98*
11	2.3	0.56	3	99*

* - Effect was statistically significant.

 

Effect Parameters

Parameter (mg/L)		NOEC	EC10	EC20	EC50
Growth rate	Value	0.29	0.61	1.0	2.5
	lower 95 %-cl	-	0.56	0.93	2.4
	upper 95 %-cl	-	0.68	1.1	2.7
Yield	Value	< 0.069	0.34	0.48	0.96
	lower 95 %-cl	-	0.27	0.41	0.88
	upper 95 %-cl	-	0.42	0.57	1.1

cl = Confidence limit.

 

Individual cell densities: According to OECD 201, the factor of the biomass parameter, measured in the control between 0 and 72 h, must be at least 16. In the current test it was found to be 190. The test fulfils this validity criterion.

Individual growth rates: The coefficient of variation of the mean specific growth rate replicates in the control between 0 and 72 h was: 0.8 %. According to OECD 201, the coefficient of variation of the mean specific growth rate, measured in the control from 0 to 72 h, must not exceed 7 %. The test fulfils this validity criterion.

Section-by-section growth rate: The mean of the replicate coefficients of variation for section-by-section growth rate in the control was: 9.9 %. According to OECD 201, the mean coefficient of variation, measured in the control from 0 to 72 h, must not be higher than 35 %. The test fulfils this validity criterion.

Statistics: Growth Rate

Shapiro-Wilk´s Test on Normal Distribution

Treatment [mg/L]	Mean	s	n
Control	1.749	0.0139	6
0.069	1.702	0.0178	3
0.29	1.710	0.0504	3
1.0	1.522	0.0297	3
3.5	0.481	0.0330	3
11	0.393	0.0595	3

Number of residuals = 21

Shapiro-Wilk’s W = 0.942

P(W) = 0.234

P(W) is greater than the selected significance level of 0.01; thus treatment data do not significantly deviate from normal distribution.

Normality check passed (p > 0.01)

 

Levene´s Test on Variance Homogeneity (with Residuals)

Source	SS	df	MSS	F	p(F)
Treatment	0.00305	5	0.00061	3.357	0.031
Residuals	0.00272	15	0.00018
Total	0.00580	20

The Levene test indicates variance homogeneity (p > 0.010).

Variance homogeneity check passed (p > 0.01).

Normal-distribution and variance-homogeneity requirements are fulfilled.

A parametric multiple test is advisable.

 

Determination of NOEC

Trend analysis by Contrasts (Monotonicity of Concentration/Response)

Trend	Psi	s(psi)	df	t	p(t)
Linear	10.62866	0.15044	15	70.649	<0.001
Quadratic	4.40431	0.16776	15	26.254	<0.001

The linear trend is significant (p <= 0.05)

The quadratic trend is significant (p <= 0.05)

The analysis of contrasts revealed a linear trend, thus the selected Williams test was performed.

 

Williams Multiple Sequential t-Test Procedure

Treatment [mg/L]	Mean	s	df	LhM	%MDD	t	t*	Sign.
Control	1.749	0.0344
0.069	1.702	0.0344	15	1.706	-2.4	-1.75	-1.75	-
0.29	1.710	0.0344	15	1.706	-2.5	-1.75	-1.83	-
1.0	1.522	0.0344	15	1.522	-2.6	-9.31	-1.85	+
3.5	0.481	0.0344	15	0.481	-2.6	-52.17	-1.86	+
11	0.393	0.0344	15	0.393	-2.6	-55.78	-1.87	+

+: significant;

-: non- significant

A NOEC of 0.290 mg/L is suggested by the program.

 

Statistics: Yield

Shapiro-Wilk´s Test on Normal Distribution

Treatment [mg/L]	Mean	s	n
Control	189.0	7.93	6
0.069	164.3	8.73	3
0.290	169.5	24.66	3
1.000	95.6	8.52	3
3.500	3.2	0.41	3
11.000	2.3	0.56	3

Number of residuals = 21

Shapiro-Wilk’s W = 0.942

P(W) = 0.106

P(W) is greater than the selected significance level of 0.01; thus treatment data do not significantly deviate from normal distribution.

Normality check passed (p > 0.01)

 

Levene´s Test on Variance Homogeneity (with Residuals)

Source	SS	df	MSS	F	p(F)
Treatment	682.706	5	136.541	8.534	< 0.001
Residuals	239.986	15	15.999
Total	922.690	20

The Levene test indicates variance heterogeneity (p <=0.010)

Variance homogeneity check failed. Nonetheless, the user-selected multiple test is performed.

 

Determination of NOEC

Non-parametric Trend analysis by Contrasts (Monotonicity of Concentration/Response)

Trend	Psi	Var(psi)	df	Chi²	p(Chi²)
Linear	-107	737.917	1	15.515	<0.001
Quadratic	-5	917.583	1	0.027	0.869

The linear trend is significant (p <= 0.05)

The quadratic trend is not significant (p >0.05)

The analysis of contrasts revealed a linear trend, thus the selected SD Jonckheere-Terpstra test was performed.

 

Step-Down Jonckheere-Terpstra Test Procedure

Treatment. [mg/L]	Mean	Med	n	J	J*	p(J)	Sign.
Control	189.0	188.0	6
0.069	164.3	167.0	3	18.0		0.0120	+
0.29	169.5	180.3	3	27.0	2.030	0.0210	+
1.0	95.6	96.5	3	63.0	3.280	<0.001	+
3.5	3.2	3.5	3	108.0	4.220	<0.001	+
11	2.3	2.4	3	162.0	5.000	<0.001	+

+: significant

-: non-significant

The NOEC is lower than 0.069 mg/L.

 

EC-Values

Parameters of the Probit Analysis (Growth Rate)

Parameter	Value
Computation runs:	12.00000
Slope b:	2.08320
Intercept a:	-0.84053
Variance of b:	1.78347
Goodness of Fit
Chi²:	0.70253
Degrees of freedom:	13.00000
p(Chi²):	1.00000
Log EC50:	0.40348
SE Log EC50:	0.21898
g-Criterion:	0.00387
Residual Variance (Chi²/df):	0.05404
r²:	0.77600
F:	45.02700
p(F) (df: 1;13):	<0.001

 

Parameters of the Probit Analysis (Yield)

Parameter	Value
Computation runs:	10.00000
Slope b:	2.79014
Intercept a:	0.04514
Variance of b:	5.50847
Goodness of Fit
Chi²:	0.64866
Degrees of freedom:	13.00000
p(Chi²):	1.00000
Log EC50:	-0.01618
SE Log EC50:	0.18847
g-Criterion:	0.03517
Residual Variance (Chi²/df):	0.04990
r²:	0.68500
F:	28.32400
p(F) (df: 1;13):	<0.001

 

Analytical Results of Test Samples

Time of Sampling (hours)	Date of Sampling	Date of Analysis*	Concentration (mg/L)		Relative to Nominal (%)	Relative to Initial (%)
			Nominal	Analysed
0	10 Dec 2018	20 Dec 2018	0	0.0014**	n.a.
			0.1	0.0965	96
			0.32	0.322	101
			1.0	1.04	104
			1.0***	1.06	106
			3.2	3.39	106
			10	10.9	109
24	11 Dec 2018	20 Dec 2018	0	0.0012**	n.a.	n.a.
			0.1	0.098	98	102
			0.32	0.330	103	102
			1.0	1.04	104	100
			1.0***	1.04	104	98
			3.2	3.5	109	103
			10	10.7	107	98
72	13 Dec 2018	20 Dec 2018	0	0.0014**	n.a.	n.a.
			0.1	0.0306	31	32
			0.32	0.218	68	68
			1.0	0.963	96	93
			1.0***	1.06	106	100
			3.2	3.52	110	104
			10	11.1	111	102

* Samples were stored in the freezer (≤-15 °C) until sample preparation on 19 Dec 2018. Due to acquisition problems the sample were analysed on 20 Dec 2018. The samples were stored at room temperature.

** Estimated value, calculated by extrapolation of the calibration curve. This is probably caused by carry over since a similar response was observed in the analytical blank.

*** Without algae.

n.a. Not applicable.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the test material inhibited growth rate significantly at TWA concentrations of 1.0 mg/L and higher.
The 72 h-EC50 for growth rate inhibition (ERC50) was 2.5 mg/L with a 95 % confidence interval ranging from 2.4 to 2.7 mg/L.
The 72 h-EC50 for yield inhibition (EYC50) was 0.96 mg/L with a 95 % confidence interval ranging from 0.88 to 1.1 mg/L.
The 72 h-NOEC for growth rate inhibition was 0.29 mg/L based on statistical significance and biological relevance.
The 72 h-NOEC for yield inhibition was below 0.069 mg/L based on statistical significance and biological relevance.

Executive summary:

The acute toxicity of the test material to algae was assessed according to OECD guideline 201 and in compliance with GLP.

The objective of the study was to evaluate the test material 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.

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 0.10, 0.32, 1.0, 3.2 and 10 mg/L. The initial algal cell density was 10^4 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 0.096, 0.32, 1.0, 3.4 and 11 mg/L, respectively. During the exposure period, the concentrations of 1.0 mg/L and higher remained stable, i.e. were at 93 - 104 % relative to initial at the end of the test. The two lowest test concentrations decreased to 32 and 68 % of initial at the end of the test

Inhibition of growth rates and yield increased with increasing concentration the test material resulting in 78 % growth rate inhibition and 99 % yield inhibition at 11 mg/L. Statistically significant inhibition of growth rates was found at test concentrations of 1.0 mg/L and higher. Statistically significant inhibition of yield was found at all test concentrations.

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

The effect parameters obtained in this study are:

NOEC (growth rate): 0.29 mg/L

EC10 (growth rate): 0.61 mg/L (95 % confidence limits of 0.56 – 0.68 mg/L)

EC20 (growth rate): 1.0 mg/L (95 % confidence limits of 0.93 – 1.1 mg/L)

EC50 (growth rate): 2.5 mg/L (95 % confidence limits of 2.4 – 2.7 mg/L)

NOEC (Yield): < 0.069 mg/L

EC10 (Yield): 0.34 mg/L (95 % confidence limits of 0.27 – 0.42 mg/L)

EC20 (Yield): 0.48 mg/L (95 % confidence limits of 0.41 – 0.57 mg/L)

EC50 (Yield): 0.96 mg/L (95 % confidence limits of 0.88 – 1.1 mg/L)

Description of key information

Key Study:

Under the conditions of the study the test material inhibited growth rate significantly at TWA concentrations of 1.0 mg/L and higher.

The 72 h-EC50 for growth rate inhibition was 2.5 mg/L with a 95 % confidence interval ranging from 2.4 to 2.7 mg/L.

The 72 h-NOEC for growth rate inhibition was 0.29 mg/L based on statistical significance and biological relevance.

Read-Across Substance:

The 72-hour EC50 for the test material to Pseudokirchneriella subcapitata, based on growth rate, was 1.1 mg/L (95 % C.I. 0.85 - 1.5 mg/L)

Key value for chemical safety assessment

EC50 for freshwater algae:

2.5 mg/L

EC10 or NOEC for freshwater algae:

0.29 mg/L

Additional information

Key Study (Vormans, 2019)

The acute toxicity of the test material to algae was assessed according to OECD guideline 201 and in compliance with GLP.

The study was assigned a reliability score of 1 in accordance with the principles for assessing data quality as set forth by Klimisch et al. (1997).

The objective of the study was to evaluate the test material 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.

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 0.10, 0.32, 1.0, 3.2 and 10 mg/L. The initial algal cell density was 10^4 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 0.096, 0.32, 1.0, 3.4 and 11 mg/L, respectively. During the exposure period, the concentrations of 1.0 mg/L and higher remained stable, i.e. were at 93 - 104 % relative to initial at the end of the test. The two lowest test concentrations decreased to 32 and 68 % of initial at the end of the test

Inhibition of growth rates and yield increased with increasing concentration the test material resulting in 78 % growth rate inhibition and 99 % yield inhibition at 11 mg/L. Statistically significant inhibition of growth rates was found at test concentrations of 1.0 mg/L and higher. Statistically significant inhibition of yield was found at all test concentrations.

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

The effect parameters obtained in this study are:

NOEC (growth rate): 0.29 mg/L

EC10 (growth rate): 0.61 mg/L (95 % confidence limits of 0.56 – 0.68 mg/L)

EC20 (growth rate): 1.0 mg/L (95 % confidence limits of 0.93 – 1.1 mg/L)

EC50 (growth rate): 2.5 mg/L (95 % confidence limits of 2.4 – 2.7 mg/L)

NOEC (Yield): < 0.069 mg/L

EC10 (Yield): 0.34 mg/L (95 % confidence limits of 0.27 – 0.42 mg/L)

EC20 (Yield): 0.48 mg/L (95 % confidence limits of 0.41 – 0.57 mg/L)

EC50 (Yield): 0.96 mg/L (95 % confidence limits of 0.88 – 1.1 mg/L)

Read-Across Substance (Vryenhoef, 2013):

The toxicity of the test material to aquatic algae was investigated in a GLP study which was conducted in accordance with standardised guidelines OECD 201 and EU Method C.3. The study was assigned a reliability score of 1 in accordance with the principles for assessing data quality as set forth by Klimisch et al. (1997).

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 89 to 96 % of nominal and so the results are based on nominal test concentrations only.

Under the conditions of the study, the 72 hour EC50, based on growth rate, was determined to be 1.1 mg/L. The No Observed Effect Concentration, based on growth rate, was determined to be 0.10 mg/L.