2-hydroxyethanesulphonic acid, compound with 4,4'-[hexane-1,6-diylbis(oxy)]bis[benzenecarboxamidine] (2:1)

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May-June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA Ecological Effects Test Guidelines, OCSPP 850.5400, Algal Toxicity, Tiers I and II

Version / remarks:

EPA 712-C-96-164, April 1996.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxyethanesulphonic acid, compound with 4,4'-[hexane-1,6-diylbis(oxy)]bis[benzenecarboxamidine] (2:1)

EC Number:

211-533-5

EC Name:

2-hydroxyethanesulphonic acid, compound with 4,4'-[hexane-1,6-diylbis(oxy)]bis[benzenecarboxamidine] (2:1)

Cas Number:

659-40-5

Molecular formula:

C20H26N4O2.2C2H6O4S

IUPAC Name:

4-[6-(4-carbamimidoylphenoxy)hexoxy]benzenecarboximidamide;2-hydroxyethanesulfonic acid

Test material form:

solid: particulate/powder

Details on test material:

Appearance: White to slightly yellow powder

Specific details on test material used for the study:

Batch 42964
Expiry date: 31 January 2019
Storage conditions: Controlled room temperature (15-25ºC, below 70 RH%), protected from humidity (tight closed container)

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

yes

Details on test solutions:

A stock solution with a concentration of 100 mg/L (nominal) was prepared with Test Item and test medium (OECD medium) by direct addition with mixing and using approximately 1-minute ultra-sonication. The test solutions were prepared by appropriate dilution of this stock solution just before introduction of the Algae (start of experiment, Day 0), distributed into test vessels prior to introduction of algae.

Test organisms

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of Citoxlab Hungary Ltd.
Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.
Breeding conditions: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.
The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Study design

Test type:

other: Volumes of 100 mL algal suspension per replicate in 250 mL Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension.

Water media type:

other: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.

Total exposure duration:

72 h

Test conditions

Test temperature:

Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was 22.0-22.9°C measured in the flask and were in the range of 21.1 and 23.0°C measured within the climate chamber.

pH:

The pH was checked at the beginning and at the end of the test, in the control and each concentration. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.78 – 8.70 during the experiment.

Nominal and measured concentrations:

Because toxic response was observed during the preliminary range-finding tests, 5 test concentrations in a geometric series (factor 1.8) and 1 Control was tested in the Main experiment.
The concentrations in the Main experiment were based on the results of the preliminary range-finding tests and were: 0.010, 0.017, 0.031, 0.056 and 0.1 mg/L (nominal).

Details on test conditions:

The algal culture flasks were continuously illuminated. The light intensity at the position occupied by algal culture flasks during the test was about 7594 lux (equivalent to ~102.6 μE/m2/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.059 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.028 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.029 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Statistical Analysis
The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period.
The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
The ErC50, EbC50 and EyC50 values of the Test Item and their confidence limits were calculated using Probit analysis by TOXSTAT software. Statistical comparisons of biomass, average specific growth rates and yield in controls and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software.
For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test.

Results with reference substance (positive control):

For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions. The date of the last study (Study Code: 17/367-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 04–07 December 2017.
The 72h ErC 50: 0.88 mg/L, (95 % confidence limits: 0.81 – 0.96 mg/L)
The 72h EbC 50: 0.63 mg/L, (95 % confidence limits: 0.58 – 0.69 mg/L)
The 72h EyC 50: 0.53 mg/L, (95 % confidence limits: 0.49 – 0.58 mg/L)
These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).

Reported statistics and error estimates:

The following deviation from the Study Plan was recorded during the study:
1) The Only the LOQ is reported in the Analytical Report instead of the LOQ and LOD together due to technical and calculation reasons.
This deviation was considered not to have adversely affecting the results or integrity of the study.

VALIDITY
The cell density in the control cultures increased by the factor of 70.17 within three days.
The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 8.23%.
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.86 %.
All validity criteria were met; therefore, the study can be considered as valid.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

ErC50 72h = 0.0588 mg/L
EyC50 72h = 0.0276 mg/L
EbC50 72h = 0.0295 mg/L

Executive summary:

The effect of Test Item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours.

Toxic response was observed during the preliminary range-finding test, therefore 5 test concentrations in a geometric series with a separation factor of 1.8 and 1 untreated Control were tested in the Main experiment.

The following nominal concentrations were tested: 0.010, 0.017, 0.031, 0.056 and 0.1 mg/L (nominal). The corresponding measured geometric mean and calculated Test Item concentrations were: 0.0060, 0.0109, 0.0198, 0.0362 and 0.0659 mg/L. The biological results are based on the measured and calculated concentrations.

The test design included 3 replicates at each test concentration and 6 replicates for the untreated Controls.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software. The ErC50, EbC50 and EyC50 values of the Test Item and their confidence limits were calculated using Probit analysis by TOXSTAT software.

In conclusion, under the conditions of this study, the calculated endpoints for the effect of the Test Item were the following:

Parameter (0-72 h)       Growth rate (r) [mg/L]       Yield (y) [mg/L]             Biomass (b) [mg/L]

EC50                            0.0588                                   0.0276                            0.0295

95 % conf. limits       0.0506 – 0.0684                    0.0252 – 0.0302              0.0268 – 0.0325

NOEC                      0.0109                                          0.0109                             0.0109

LOEC                      0.0198                                          0.0198                              0.0198