Lithium dihydrogen 5-sulphonatoisophthalate

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:

July-December 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

No deviations in standard operating procedures, but minor deviations in the protocols, which did not adversely affect the study integrity. These deviations are listed in the results section.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium hydrogen-5-sulphoisophthalate

EC Number:

228-845-2

EC Name:

Sodium hydrogen-5-sulphoisophthalate

Cas Number:

6362-79-4

Molecular formula:

C8H6O7S.Na

IUPAC Name:

Sodium 3,5-dicarboxybenzenesulfonate

Constituent 2

Reference substance name:

5-(Sodiosulfo)isophthalic acid

IUPAC Name:

5-(Sodiosulfo)isophthalic acid

Test material form:

solid: particulate/powder

Details on test material:

5-(Sodiosulfo)isophthalic acid, sodium salt

Molecular formula C8H5O7S.Na
Molecular weight 268.18
CAS Number 6362-79-4
Description White powder
Batch 6F21174000
Purity/Composition 99.4%
Test substance storage At room temperature in the dark
Stability under storage conditions Stable
Expiry date 02 December 2013

Volatile No
Stability in water Not indicated
Solubility in water 0.345 g/mL

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Singular samples for possible analysis were taken from all test concentrations and the control according to the schedule below.

Frequency: All test groups at t=0 h and t=24 h. At t=72 h only the control, 10 and 100 mg/l.
Volume: 1.8 ml
Storage: Samples were stored in a freezer until analysis.

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 demonstrated by running a test vessel at the highest substance concentration 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, singular reserve samples of 1.8 ml were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

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:

Pre-culture: 4 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:

Preparation of test solutions:

The batch of 5-(Sodiosulfo)isophthalic Acid tested was a white powder with a purity of 99.4% and the substance was completely soluble in test medium at the concentrations tested.
Preparation of test solutions for the limit test started with the highest test concentration of 100 mg/l. No special treatment other than careful mixing was necessary in order to fully dissolve this concentration in test medium. The pH of this solution was adjusted from 4.7 to 6.0 using NaOH (1 M, Merck, Darmstadt, Germany). The lower test concentrations for the combined range-finding test were prepared by subsequent dilutions in test medium. The final test solutions were all clear and colourless.
After preparation, volumes of 50 ml were added to each replicate test concentration.

Test organisms

Test organisms (species):

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

Details on test organisms:

Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.

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.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

Study was a combined limit/range-finding test.

Post exposure observation period:

None

Test conditions

Test temperature:

Measured continuously in a temperature control vessel.

pH:

Recorded at the beginning and at the end of the test.
Within the range of 6.0-9.0
The pH of the solutions should preferably not deviate by more than 1.5 units during the test.

Nominal and measured concentrations:

Nominal: 0.1, 1.0, 10 and 100 mg/l, the regulatory limit concentration.
Measured: In samples incubated with algae at the nominal 100 mg/l concentration, actual measured concentrations were 91.4 (0 h), 93.8 (24 h) and 96.5 (72 h).

Details on test conditions:

5-(Sodiosulfo)isophthalic Acid 0.1, 1.0, 10 and 100 mg/l, the regulatory limit concentration.

Controls Test medium without test substance or other additives.

Replicates 6 replicates of the control and 100 mg/l,
3 replicates of 0.10, 1.0 and 10 mg/l,
1 or 2 extra replicates of each test group for sampling purposes,
1 or 2 replicates of each test concentration without algae.

Test duration: 72 hours
Test type: Static
Test vessels: 100 ml, all-glass, containing 50 ml of test solution
Medium: M2
Cell density: An initial cell density of 1 x 104 cells/ml.
Illumination: Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 93 to 98 μE.m-2.s-1.
Incubation: Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

pH was measured at the beginning and at the end of the test.
The pH of the solutions should preferably not deviate by more than 1.5 units during the test.

Temperature of medium was measured continuously in a temperature control vessel.

Appearance of the cells: At the end of the test microscopic observations were performed on the limit concentration of 100 mg/l to observe for any abnormal appearance of the algae.

Recording of cell densities: 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 720 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions.

Electronic data capture:
Observations/measurements in the study were recorded electronically using the following programme(s):
- Shimadzu Spectrophotometer UV-1800 including UVProbe 2.33 software (Shimadzu, Kyoto, Japan): Algal cell density.
- REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA): Temperature.

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.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of yield

Effect concentrations 3

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Acceptability of the test:
1. In the controls, cell density increased by an average factor of > 16 within three days.
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35%.
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7%.

List of protocol deviations:
1. Temperature of test solutions at study initiation was 20.5°C, which is just below the optimal range of 21-24°C.
Evaluation: Temperature during incubation was within the required range. The short and minimal lower temperature just before incubation does not affect the study result.
2. The vessels containing solutions of 0.10 and 1.0 mg/l all became turbid during testing and it was decided to omit these vessels from the test.
Evaluation: Results of the test were based on the remaining test concentrations, being the control, 10 and 100 mg/l. This proved sufficient as no adverse effects were observed on algal growth.
The study integrity was not adversely affected by the deviations.

Measured test substance concentrations:
Analyses of the samples taken from 100 mg/l showed that the measured concentration was stable and in agreement with nominal throughout the test period.

Note that analyses of the samples taken from the solution incubated without algae showed that concentrations were in agreement with nominal during the first 24 hours but unexpectedly decreased to 10 mg/l after 72 hours of exposure. The cause of this decrease was unknown. This however does not affect the result of the study as this will be based on the result obtained in the solutions incubated with algae.

Mean cell densities:
Table 1 shows mean cell densities measured at 24-hour intervals at the different concentrations of 5-(Sodiosulfo)isophthalic Acid. The respective growth curves are shown in Figure 1 (see APPENDIX 1 for the cell densities per replicate). Note that vessels containing 0.10 and 1.0 mg/l were turbid and omitted from the test results.

The pH was within the limits prescribed by the protocol (6.0-9.0, preferably not varying by more than 1.5 unit). The temperature of the test medium was 20.5°C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 21.7 and 22.3°C.

Any other information on results incl. tables

Percentage reduction of growth rate (total test period) and percentage inhibition of yield
Concentration 5-(Sodiosulfo) isophthalic Acid (mg/l) Mean growth rate   Yield (0-72 h)   μ (0-72 h)   Reduction (%)   x10^4cells/ml   Inhibition (%)   Control 0.05752     62.03   10 0.06048   -53.8   77.07 -24.2 100 0.06048   -54.5   77.12 -24.3

Percentage reduction of growth rate at different time intervals
Concentration 5-(Sodiosulfo) isophthalic Acid (mg/l) Mean growth rate   μ (0-24 h)   Reduction (%)   μ (24-48 h)   reduction (%)   μ (48-72 h)   reduction (%)   Control 0.03577     0.00800   0.05680     10 0.05503   -53.8   0.06424 19.7 0.06218   -9.5 100 0.05525   -54.5   0.06191 22.63 0.06427   -13.2

pH Levels

Concentration 5-(Sodiosulfo) isophthalic Acid (mg/l)	Exposure time (hours)
	0	72
Control	8.0	7.7
10	7.9	7.7
100	6.3	7.2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, no reduction of growth rate or inhibition of yield was recorded at any of the concentrations of 5-(Sodiosulfo)isophthalic Acid tested.

The EC50 for both growth rate reduction (ERC50: 0-72h) and yield inhibition (EYC50: 0-72h) exceeded 100 mg/l.

The NOEC for growth rate reduction and yield inhibition was >=100 mg/l.

Executive summary:

A limit test was combined with a range-finding test. Six replicates of exponentially growing algal cultures were exposed to a control and to a nominal 5-(Sodiosulfo)isophthalic Acid concentration of 100 mg/l in the limit test. Three replicates per test group were exposed to 0.10, 1.0 and 10 mg/l in the combined range-finding test. The initial algal cell density was 104 cells/ml and the total test period was 72 hours. Samples for analytical confirmation of actual exposure concentrations were taken from all test groups at the start, after 24 and 72 hours of exposure.

Analyses of the samples taken from 100 mg/l showed that the measured concentration was stable and in agreement with nominal throughout the test period.

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

No reduction of growth rate or inhibition of yield was recorded at any of the concentrations of 5-(Sodiosulfo)isophthalic Acid tested.

The EC50 for both growth rate reduction (ERC50: 0-72h) and yield inhibition (EYC50: 0-72h) exceeded 100 mg/l. The NOEC for growth rate reduction and yield inhibition was >= 100 mg/l.