5,5'-diisopropyl-2,2'-dimethylbiphenyl-4,4'-diyl dihypoiodite

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:

20 April 2018 to 12 June 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:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 23 (Guidance document on aquatic toxicity testing of difficult substances and mixtures)

Version / remarks:

2000

GLP compliance:

yes

Specific details on test material used for the study:

- The water solubility at 20 °C was determined to be < 0.08 mg/L, using the column elution method

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the filter containing the undissolved residue was kept for possible analysis.

> Schedule:
- Frequency: at t=0 h, t=24 h and t=72 h.
- Volume: 2.0 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 the WAF prepared at a loading rate of 4.0 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 2.0 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:

The batch of test material tested was not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test material.
Preparation of test solutions started with loading rates individually prepared in the range of 0.20 to 100 mg/L. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test material in medium. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by filtration through a 0.45 µm membrane filter (RC55, Whatman, in the combined limit/range-finding test and PES450, Supor, in the final test) and used as test concentrations. All test solutions were clear and colourless 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:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum (at test initiation):

CULTIVATION
- 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 (light intensity: 60 - 120 µE/m²/s when measured in the photosynthetically effective wavelength range of 400 - 700 nm) in a climate room at a temperature of 21-24°C.
- 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) with the following composition: NaNO3 (500 mg/L), K2HPO4 (39.5 mg/L), MgSO4.7H2O (75 mg/L), Na2CO3 (20 mg/L), C6H8O7.H2O (6 mg/L), NH4NO3 (330 mg/L), CaCl2.2H2O (35 mg/L), C6H5FeO7.xH2O (6 mg/L), H3BO3 (2.9 mg/L), MnCl2.4H2O (1.81 mg/L), ZnCl2 (0.11 mg/L), CuSO4.5H2O (0.08 mg/L), (NH4)6Mo7O24.4H2O (0.018 mg/L)
- Pre-culture: Three 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.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

22 - 23 °C

pH:

8.0 - 8.5

Nominal and measured concentrations:

WAFs individually prepared at loading rates of 0.20, 0.80, 4.0, 20 and 100 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass with aluminium caps, perforated for ventilation
- Fill volume: 50 mL
- Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
- Initial cells density: 1 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- 1 extra replicate of each test group for sampling purposes after 24 hours of exposure
- 1 or 2 replicates of each test concentration without algae.

GROWTH MEDIUM
- Standard medium used: yes - M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition: NH4Cl (15 mg/L), MgCl2.6H2O (12 mg/L), CaCl2.2H2O (18 mg/L), MgSO4.7H2O (15 mg/L), KH2PO4 (1.6 mg/L), FeCl3.6H2O (64 µg/L), Na2EDTA.2H2O (100 µg/L), H3BO3 (185 µg/L), MnCl2.4H2O (415 µg/L), ZnCl2 (3 µg/L), CoCl2.6H2O (1.5 µg/L), CuCl2.2H2O (0.01 µg/L), Na2MoO4.2H2O (7 µg/L), NaHCO3 (50 mg/L), Hardness (Ca+Mg) (0.24 mmol/L (24 mg CaCO3/L); pH 8.1 ± 0.2

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test, for all test concentrations and the control. The temperature of the medium was measured continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Photoperiod: Continuous (using TLD-lamps)
- Light intensity and quality: 87 to 90 µE.m^-2.s^-1

EFFECT PARAMETERS MEASURED
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.
At the end of the final test microscopic observations were performed on the WAF prepared at a loading rate of 0.80 mg/L to observe for any abnormal appearance of the algae compared to the control.


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1.0 and 10 mg/L
- Results used to determine the conditions for the definitive study: yes

DATA HANDLING
- Calibration Curve
Quantification of cell densities was based on a calibration curve. Cell density was plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of an algal suspension with different cell densities. The calibration curve was composed using linear regression. The software automatically calculates the cell densities based on this curve for the spectrophotometric measurements at the various points in time during the test period.

- Comparison of Average Growth Rates
The average specific growth rate for a specific period is calculated as the logarithmic increase in the biomass from the equation for each single vessel of controls and treatments:
µi-j = (ln Xj - ln Xi) / (tj - ti)
where: µi-j = the average specific growth rate from time i to j (day^-1)
Xi = the biomass at time i
Xj = the biomass at time j

The average growth rate at each test material concentration is then compared with the control value and the percentage inhibition in growth rate is calculated:
%Ir = (µC - µT) / µC x 100
where: %Ir = percent inhibition in average specific growth rate
µC = mean value for average specific growth rate in the control group
µT = average specific growth rate for the treatment replicate

- Yield
The percent inhibition in yield is calculated for each treatment replicate as follows:
%Ir = (YC - YT) / YC x 100
where: %Iy = percent inhibition of yield
YC = mean value for yield in the control group
YT = value for yield for the treatment replicate

ANALYSIS
For determination of the NOELR the approach recommended in the OECD guideline 201 was 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 or inhibition of yield (Williams Multiple Sequential t-Test, α=0.05, one-sided, smaller).
The EC50 for growth rate could not be determined since the recorded effects were below 50%. In addition, ECx-values for growth rate and yield could not be calculated since no quantifiable test material responses were measured at majority of the test concentrations. Instead, ELx-values were determined.
The EL10 for growth rate, EL10 for yield and the EL20 for yield could not be calculated using any regression method. The test concentration at which growth rate inhibition was closest to 10% is reported as the EL10, however, should be interpreted as an indication rather than a true value.
Calculation of the EL20 for growth rate and EL50 for yield 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 loading rates of the test material.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

ACCEPTABILITY OF THE TEST
For a test to be valid, the following must be achieved:
- In the control, cell density should increase by an average factor of at least 16 within the exposure period
- The mean coefficient of variation for section-by-section specific growth rates in the control cultures must not exceed 35 %
- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7 %

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.5 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks:

(biological relevance)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks:

statistical significance

Details on results:

RANGE-FINDING TEST
At the end of the test, biologically relevant growth rate and yield inhibition (i.e. >10 %) was measured at all test concentrations. No increase of growth rate or yield inhibition was recorded between the WAFs prepared at 10 and 100 mg/L which indicates a flat concentration-response curve.
Based on these results, samples taken from all test concentrations were analysed. No detectable test material response was measured at any of the test concentrations throughout the test. Consequently, the filter containing the undissolved fraction of the test material removed during preparation of test solutions was analysed. Qualitative analysis showed presence of the test material in the filter residue which demonstrated that the test material was used during preparation of test solutions.
All test conditions were maintained within the limits prescribed by the study plan.

FINAL TEST
MEASURED TEST MATERIAL CONCENTRATIONS
Samples taken from all test concentrations and the control were analysed. In general, no detectable test material response was observed in the control and at the three lowest test concentrations throughout the test. In several samples, only a small response at the retention time of the test material was recorded which was below the limit of quantification (LOQ; 0.1 mg/L). At the WAF prepared at a loading rate of 20 mg/L, an initial concentration of 0.15 mg/L could be quantified, which decreased below the LOQ already after 24 hours of exposure. In the sample taken from the highest test concentration at the start of the test, the measured concentration was 0.50 mg/L and remained stable (i.e. 80-84 % relative to nominal) throughout the test.
Actual exposure concentrations in the samples taken from the WAF prepared at 4.0 mg/L, with and without algae, could not be quantified as the responses were below the LOQ. Hence, it is unknown whether the presence of algae affected the test material concentrations in test medium. Since no test material concentration was measured in the solution without algae throughout the test, it can be assumed that at least other mechanisms than metabolism or binding to algae are responsible for the absent or decreasing test material concentrations over time. Nevertheless, it can be stated that testing was ultimately performed at the maximum soluble concentration of test material in test medium.
Based on the obtained results, effect parameters were based on loading rates (e.g. NOELR, EL10, EL20 and EL50). Since actual exposure concentrations could be quantified in the samples taken from the highest test concentration, the EC50 for growth rate and yield could be expressed in terms of the initially measured concentration.

INHIBITION OF GROWTH AND INHIBITION OF YIELD
A concentration-related increase of growth rate and yield inhibition was recorded at all test concentrations resulting in 29 % growth rate and 81 % yield inhibition at the highest test concentration. Statistically significant growth rate and yield inhibition was found at all test concentrations. Growth rate inhibition recorded at the WAF prepared at 0.20 mg/L was considered to be biologically not relevant as the observed inhibition was below 10%. The NOELR based on biological relevance was thus set at 0.20 mg/L.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the WAF prepared at a loading rate of 0.80 mg/L when compared to the control.

Results with reference substance (positive control):

The objective of the study was to evaluate Potassium dichromate for its ability to generate toxic effects in Pseudokirchneriella subcapitata uring an exposure period of 72 hours and, if possible, to determine the EC50 for inhibition of both growth rate and yield. The study procedures were based on the standard guideline OECD 201.
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.
Potassium dichromate inhibited growth rate and yield of this fresh water algae species at nominal concentrations of 0.18 mg/L and higher.
The EC50 for growth rate inhibition (72h-ERC50) was 0.90 mg/L with a 95 % confidence interval ranging from 0.88 to 0.93 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (72h-EYC50) was 0.30 mg/L with a 95 % confidence interval ranging from 0.29 to 0.31 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the test material inhibited growth rate and yield of the fresh water algae species significantly at Water Accommodated Fractions (WAFs) prepared at individual loading rates of 0.20 mg/L and higher.
The 72h-EC50 for growth rate inhibition (ERC50) was beyond the measured concentration of 0.50 mg/L, being considered as the maximum soluble concentration of test material in test medium.
The 72h-NOELR for growth rate inhibition was below 0.20 mg/L based on statistical significance.
The 72h-NOELR for growth rate inhibition was 0.20 mg/L based on biological relevance.

Executive summary:

The toxicity of the test material to freshwater algae was investigated in a study which was conducted in accordance with the standardised guideline OECD 201, under GLP conditions.

The test material was not completely soluble in test medium at the loading rates initially prepared. Water Accommodated Fractions (WAFs) were individually prepared at loading rates in the range of 0.20 to 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 prepared at loading rates of 0.20, 0.80, 4.0, 20 and 100 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.

A concentration-related increase of growth rate and yield inhibition was recorded at all test concentrations resulting in 29 % growth rate and 81 % yield inhibition at the highest test concentration. Statistically significant growth rate and yield inhibition was found at all test concentrations. Growth rate inhibition recorded at the WAF prepared at 0.20 mg/L was considered to be biologically not relevant as the observed inhibition was below 10 %.

Samples taken from all test concentrations and the control were analysed. In general, no detectable test material response was observed in the control and at the three lowest test concentrations throughout the test. In several samples, only a small response at the retention time of the test material was recorded which was below the limit of quantification (LOQ; 0.1 mg/L). At the WAF prepared at a loading rate of 20 mg/L, an initial concentration of 0.15 mg/L could be quantified, which decreased below the LOQ already after 24 hours of exposure. In the sample taken from the highest test concentration at the start of the test, the measured concentration was 0.50 mg/L and remained stable (i.e. 80-84 % relative to nominal) throughout the test.

Based on the obtained results, effect parameters were based on loading rates (e.g. NOELR, EL10, EL20 and EL50). The EC50 for growth rate and yield was expressed in terms of the initially measured concentration.

Under the conditions of the study the test material inhibited growth rate and yield of the fresh water algae species significantly at Water Accommodated Fractions (WAFs) prepared at individual loading rates of 0.20 mg/L and higher.

The 72h-EC50 for growth rate inhibition (ERC50) was beyond the measured concentration of 0.50 mg/L, being considered as the maximum soluble concentration of test material in test medium.

The 72h-NOELR for growth rate inhibition was below 0.20 mg/L based on statistical significance.

The 72h-NOELR for growth rate inhibition was 0.20 mg/L based on biological relevance.

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

Description of key information

Under the conditions of the study the test material inhibited growth rate and yield of the fresh water algae species significantly at Water Accommodated Fractions (WAFs) prepared at individual loading rates of 0.20 mg/L and higher.

The 72h-EC50 for growth rate inhibition (ERC50) was beyond the measured concentration of 0.50 mg/L, being considered as the maximum soluble concentration of test material in test medium.

The 72h-NOELR for growth rate inhibition was below 0.20 mg/L based on statistical significance.

The 72h-NOELR for growth rate inhibition was 0.20 mg/L based on biological relevance.

Additional information

The toxicity of the test material to freshwater algae was investigated in a study which was conducted in accordance with the standardised guideline OECD 201, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was not completely soluble in test medium at the loading rates initially prepared. Water Accommodated Fractions (WAFs) were individually prepared at loading rates in the range of 0.20 to 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 prepared at loading rates of 0.20, 0.80, 4.0, 20 and 100 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.

A concentration-related increase of growth rate and yield inhibition was recorded at all test concentrations resulting in 29 % growth rate and 81 % yield inhibition at the highest test concentration. Statistically significant growth rate and yield inhibition was found at all test concentrations. Growth rate inhibition recorded at the WAF prepared at 0.20 mg/L was considered to be biologically not relevant as the observed inhibition was below 10 %.

Samples taken from all test concentrations and the control were analysed. In general, no detectable test material response was observed in the control and at the three lowest test concentrations throughout the test. In several samples, only a small response at the retention time of the test material was recorded which was below the limit of quantification (LOQ; 0.1 mg/L). At the WAF prepared at a loading rate of 20 mg/L, an initial concentration of 0.15 mg/L could be quantified, which decreased below the LOQ already after 24 hours of exposure. In the sample taken from the highest test concentration at the start of the test, the measured concentration was 0.50 mg/L and remained stable (i.e. 80-84 % relative to nominal) throughout the test.

Based on the obtained results, effect parameters were based on loading rates (e.g. NOELR, EL10, EL20 and EL50). The EC50 for growth rate and yield was expressed in terms of the initially measured concentration.

Under the conditions of the study the test material inhibited growth rate and yield of the fresh water algae species significantly at Water Accommodated Fractions (WAFs) prepared at individual loading rates of 0.20 mg/L and higher.

The 72h-EC50 for growth rate inhibition (ERC50) was beyond the measured concentration of 0.50 mg/L, being considered as the maximum soluble concentration of test material in test medium.

The 72h-NOELR for growth rate inhibition was below 0.20 mg/L based on statistical significance.

The 72h-NOELR for growth rate inhibition was 0.20 mg/L based on biological relevance.

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