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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Analytical Support: 19 September 2017 and 21 May 2018, Definitive Assay: 05 May 2018 and 08 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
adopted 23 March 2006; Annex 5 corrected 28 July 2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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.
Vehicle:
yes
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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23.1 – 23.5°C
pH:
7.13-8.45
Nominal and measured concentrations:
Concentrations in [mg/L]
--------------------------------------------------------------------------------------
Nominal Measured concentration
--------------------------------------------------------------------------------------
0 h 72 h (old media) Geometric mean
--------------------------------------------------------------------------------------
10 12 6.45 8.8
18 20.7 7.63 13
32 36.4 15.9 24
56 60.4 32.2 46
100 106 58.2 79
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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 3-hydroxy-4-oxo-3,4-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 10, 18, 32, 56 and 100 mg/L.

Preparation of Test Medium
At the start of the test, 100.01 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. Dilutions were prepared, from the 100 mg/L test concentration, in EC media to give the 56, 32, 18 and 10 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 10^4 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
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
26 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: degradation product
Basis for effect:
biomass
Key result
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
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
27 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: degradation product
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
18 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other:
Remarks:
degradation product
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: degradation product
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
7.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: degradation product
Basis for effect:
other: Yield
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
Validity criteria fulfilled:
yes
Conclusions:
Based on geometric mean measured concentrations, the 72-hour EyC50 and the 0-72 hour EbC50 and ErC50 values were calculated to be 27, 26 and 68 mg/L, respectively. The corresponding EC10 values for yield, biomass and specific growth rate after 72 hours were 7.4, 9.0, and 18 mg/L.

Description of key information

The 72 -hour EC50 value for growth rate was determined to be 68 mg/L and the 72 -h EC10 value for growth rate was determined to be 18 mg/L in study conducted according to OECD Guideline 201.

Key value for chemical safety assessment

EC50 for freshwater algae:
68 mg/L
EC10 or NOEC for freshwater algae:
18 mg/L

Additional information