Sodium 6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate

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:

Analytical Support: 20 September 2017 and 04 June 2018, Definitive Assay: 29 May 2018 and 01 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

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Sodium 6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate

EC Number:

220-189-5

EC Name:

Sodium 6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate

Cas Number:

2657-00-3

Molecular formula:

C10H6N2O4S.Na

IUPAC Name:

sodium 6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

At the start of the test (0 hours), 10 mL samples of freshly prepared test media were taken from the control and each test media preparation flask for chemical analysis.
At 72 hours, inoculated replicate test vessels at each treatment level were pooled and sampled. Samples (10 mL) of each were taken for chemical analysis.

Test solutions

Vehicle:

yes

Test organisms

Test organisms (species):

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

Details on test organisms:

The axenic strain of Pseudokirchneriella subcapitata (CCAP 278/4) was obtained from a concentrated liquid slope culture from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Oban, UK.
On receipt from the CCAP, the slope culture was stored in the fridge at 2 8 °C. A typical shelf life for each slope was 8 months, after which the slopes were discarded.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

23.1 – 23.5°C

pH:

7.47-9.78

Nominal and measured concentrations:

Concentrations in [mg/L]
--------------------------------------------------------------------------------------
Nominal Measured concentration
--------------------------------------------------------------------------------------
0 h 72 h (old media) Geometric mean
--------------------------------------------------------------------------------------
6.25 6.87 5.89 6.36
12.5 13.1 11.5 12.3
25 25.9 15.0 20
50 52.9 42.2 47
100 111 82.1 96
--------------------------------------------------------------------------------------

Details on test conditions:

Test Procedures

Range-finding Test
A range-finding test was conducted at nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L. A control group was also included. Duplicate test vessels were prepared for the control and each test concentration. Chemical analysis of the test preparations was not conducted during the range-finding test.

Solubility/Stability Trial
Following completion of the range-finding test, initial trials were conducted to determine the solubility and stability of the test substance in test media. Initial trials showed highly variable analytical results.
During analytical method development it was realised that the test substance was highly unstable in the test media. Further work indicated that the DT50 was <1 hour. As recommended by the OECD Series on Testing and Assessment, No. 23 (2000); (Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures), the testing was therefore conducted on the degradation product 6-hydroxy-5-oxo-5,6-dihydronaphthalene-1-sulfonic acid.
A solubility/stability trial was therefore conducted to determine the solubility and stability of the degradation product in test media over the duration of the test.
Test concentrations of 100 mg/L were prepared in duplicate in EC medium. The preparations were left under laboratory conditions for ca 2 hours to fully degrade. Samples were taken for analysis from each preparation at 0 hours. From each preparation vessels were prepared with and without algae and then left under test conditions for 72 hours after which further samples were taken for chemical analysis.

Definitive Test
Based on the results of the range-finding tests, the definitive test was conducted at nominal test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L.

Preparation of Test Medium
At the start of the test, 99.97 mg of test substance was dissolved in 1000 mL of EC medium to give the 100 mg/L test concentration. The preparation was left at room temperature for approximately 2 hours to degrade. Serial dilutions were prepared in EC media to give the 50, 25, 12.5 and 6.25 mg/L test concentrations. A control group was prepared by adding EC medium only to the control vessels.

Appearance of Test Media
The appearance, colour and behaviour of the test substance in the test media were recorded at the start and end of the test.

Test Vessel Preparation
The test vessels were sterile autoclaved glass 250 mL Erlenmeyer (conical) flasks, into which 100 mL of the appropriate control or test media was added.
Three test vessels were prepared for each test concentration as well as six control vessels (EC medium only). Each test and control vessel was inoculated with sufficient Pseudokirchneriella subcapitata cells to achieve a starting algae cell concentration of 1 x 104 cells/mL. An additional inoculated vessel was prepared for the control and each test concentration for initial water quality analysis. The shape and general condition of the algae in the culture was assessed by microscopic observations prior to use.
A media blank (EC media only) was prepared to establish background counts on each sampling occasion. Background counts were subtracted from the cell counting results for each of the inoculated test vessels. The resulting cell counts were then used to calculate the yield and the corresponding specific growth rates.

Test Vessel Sampling
At approximately 24-hour intervals after the start of the incubation period, pre determined volumes of test media were removed from each incubated test vessel, and transferred to individually identified cell counting vials. The contents of each vial were diluted to a 10 mL final volume with an electrolyte solution. The cell density of the vial contents was then determined using a particle counter (Z2 Coulter Counter®).

Environmental Conditions
All flasks were loosely-capped and incubated in a temperature and light controlled incubator. The vessels were shaken at ca 100 rpm under conditions of constant light (4440 to 8880 Lux), using florescent lights, emitting white light across the visible portion of the spectrum (400 - 700 nm).
At the start of the test, the pH of freshly prepared test media was determined. The pH in each test vessel was also determined at the end of the test.
The temperature was set within the range 21 to 24 °C. The temperature was recorded continuously using a digital temperature logger.
The light intensity within the incubator was monitored at the start and end of the test at four separate locations within the test area.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: degradation product

Basis for effect:

biomass

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

68 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: degradation product

Basis for effect:

growth rate

Details on results:

Based on geometric mean measured concentrations, the 72-hour EyC50 and ErC50 values were calculated to be 45, and 68 mg/L, respectively

Results with reference substance (positive control):

Yield LCL(mg/L)- UCL (mg/L) Area Under Curve LCL(mg/L)- UCL (mg/L) Growth Rate LCL(mg/L)- UCL (mg/L)
EC10 (mg/L) 0.0810 0.0118 – 0.773 0.0706 0.0192 – 0.650 0.791 0.349 – 1.43
EC20 (mg/L) 0.449 NA – 1.03 0.416 NA – 0.874 1.16 0.963 – 1.49
EC50 (mg/L) 1.19 0.514 – 2.01 1.21 0.504 – 1.93 1.93 1.69 – 2.44

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on geometric mean measured concentrations, the 72-hour EyC50 and ErC50 values were calculated to be 45, and 68 mg/L, respectively