Tris(N-hydroxy-N-nitrosophenylaminato-O,O')aluminium

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:

July-August 2016

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Test item name: RCX 15-116
- Batch number: 15047AE2
- Other name(s): NPAL
- Appearance/colour: solid/whitish
- Chemical name: aluminium-N-nitrosophenylhydroxylamine (NPAL)
- CAS number: 15305-07-4
- Purity analysed: 99.1 %
- Molecular weight 438.3 g/mol
- Issue date of certificate: 08 Feb 2016
- Expiry date: 30 Nov 2016
- Stability: instable after repeated contact to air and / or light, undergoes hydrolysis
- Storage conditions: room temperature, protected from light

Analytical monitoring:

yes

Details on sampling:

Analytical samples were taken and analysed in the main test at 0 hours (initial value) from fresh test solutions and at 72 hours from aged test solutions from all test concentrations, solvent control and control.

Vehicle:

yes

Details on test solutions:

Following a range-finding test with nominal test item concentrations of 0.00100, 0.0100, 0.100, 1.00 and 10.0 mg/L, solvent control and control a main test with nominal concentrations of 1.08, 3.36, 10.4, 32.3, 100 mg/L, solvent control and control was performed.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Desmodesmus subspicatus Chodat (Sphaeropleales: Scenedesmaceae) Strain: SAG 86.81, was used as the test organism from a commercial supplier (MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany). The algae were grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Stock cultures are ordered regularly from our commercial supplier.
Culture conditions were as follows:
• Illumination: continuously (approx. 4440 - 8880 lux at cell culture level or 60 – 120 µE m-2 s-1)
• Temperature: 21 - 24 °C
• Culture flasks: 100 mL Erlenmeyer flasks
• CO2 supply by shaking on a rotating shaker, approximately 105 rpm

Cells from this semi-continuous liquid stock culture were used for the test. 3 to 4 days before starting the test, the algae were held under test conditions in the exponential growth phase and in test medium to produce a pre-culture. The test enables the determination of toxic effects of water soluble substances on the cell growth of the fresh water algae Desmodesmus subspicatus within an incubation period of 72 h.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.6 – 22.4 °C

pH:

The medium used for the test was AAP-Medium (According to Annex 3 of OECD 201, composition see Appendix BAppendix B). The pH of control: was 7.44 – 7.77.

Dissolved oxygen:

No data.

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Following a range-finding test with nominal test item concentrations of 0.00100, 0.0100, 0.100, 1.00 and 10.0 mg/L, solvent control and control a main test with nominal concentrations of 1.08, 3.36, 10.4, 32.3, 100 mg/L, solvent control and control was performed. The measured initial concentration of the stock solutions ranged from 53 % to 68 % of nominal with a mean initial concentration of 64 % of nominal. Therefore toxicological endpoints were evaluated using the nominal concentrations of the test item.

Details on test conditions:

Six replicates were performed for the control as well as for the solvent control and three for each test item concentration. The test was performed in 100 mL Erlenmeyer flasks under continuous illumination at 71.2 – 85.3 µEm-2s-1 at cell culture level. The final volume in each test vessel was 50 mL.

The algae were exposed to different concentrations of the test item under defined conditions in a synthetic growth medium for a time period of 72 h. The initial cell density was adjusted to 0.5 × 104 algae per mL. By comparing the cell division under test conditions with and without the influence of the test item, an inhibition of the cell multiplication can be calculated. This inhibition is a value for toxicity. The cell numbers are determined by fluorescence detection. At 24, 48 and 72 hours from the test initiation, the number of cells in each replicate was evaluated and used to calculate effect concentrations which resulted in 50, 20 and 10 % inhibition of the cell growth rate (ErCx) and yield (EyCx). The concentration which did not cause any significant inhibition (NOEC) and the lowest observed effect concentration (LOEC) were also determined, where possible.

Potassium dichromate is tested as the toxic reference item in a separate study twice a year to confirm the sensitivity of the test organism against compounds with known effects under the test conditions. The ECx values calculated in this reference test were considered to be within an acceptable range, therefore it can be considered that the algae cells are sensitive.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate is tested as the toxic reference item in a separate study twice a year to confirm the sensitivity of the test organism against compounds with known effects under the test conditions

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

60 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

11.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

32.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.36 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

The measured initial concentration of the stock solutions ranged from 53 % to 68 % of nominal with a mean initial concentration of 64 % of nominal. Therefore toxicological endpoints were evaluated using the nominal concentrations of the test item. An inhibition of growth rate and yield was observed in all test item concentrations. At higher test item concentrations the inhibition of growth rate peaked in 62.1 % and the inhibition of yield peaked in 91.0 %. The morphology of the algae cells was observed microscopically at test end. The cells were considered normal for the control and up to and including a test item concentration of 32.3 mg/L. A distinct reduction of cells was observed at 100 mg/L test item concentration.

The EC50-value for growth rate (ErC50) was 60.0mg/L (nominal). The EC50-value for yield (EyC50) was11.5mg/L (nominal). For growth rate the NOEC was determined to be at 10.4 mg/L (nominal) and the LOEC at 32.3 mg/L. For yield the NOEC was determined to be at 3.36 mg/L and the LOEC at 10.4 mg/L (nominal).

Reported statistics and error estimates:

The statistical evaluation for the 72 hours period was performed for growth rate and yield using SAS® (2002–2010). A test for normality of the data was performed by calculating the Shapiro-Wilk statistic and the homogeneity of variance of the data was evaluated by calculating the Bartlett Test. The NOEC and LOEC were determined by using Dunnetts-t-test. The EC50-values for growth rate and yield were determined by the probit analysis following the Gompertz and normal distribution, respectively. Only concentrations within a clear dose response were used for calculations. Since no statistically significant differences were determined between the control and the solvent control the evaluations of the toxicological endpoints were based on the solvent control.

Biomass: Cell numbers, measured in the solvent controls between 0 h and 72 hours, were found to increase by a factor of 45.7 for the solvent control and 55.35 for the control, which exceeds the threshold of 16. It corresponds to a growth rate of 1.27342 d-1 for the solvent control and 1.33687 d-1 for the control, respectively.

Coefficient of Variation (section by section): The mean coefficient of variation for the section-by-section specific growth rates (hours 24 - 0, 48 - 24 and 72 - 48) in the solvent control cultures was 25 % and 13 % for the control and did not exceed 35 %.

Coefficient of Variation: (average growth): The coefficient of variation of average growth in replicate solvent control cultures was 7 % for the solvent control, 4.2 % for the control and did not exceed 7 % for the whole test period.

Validity criteria fulfilled:

yes

Conclusions:

The EC50-value for growth rate (ErC50) was 60.0mg/L (nominal). The EC50-value for yield (EyC50) was11.5mg/L (nominal). For growth rate the NOEC was determined to be at 10.4 mg/L (nominal) and the LOEC at 32.3 mg/L. For yield the NOEC was determined to be at 3.36 mg/L and the LOEC at 10.4 mg/L (nominal).

Executive summary:

The objective of the study was to assess theeffect of RCX 15-116 in a growth inhibition test using the single cell green alga Desmodesmus subspicatus with the determination of the No Observed Effect Concentration (NOEC), the Lowest Observed Effect Concentration (LOEC) and the Effect Concentrations (EC_10,20,50), where possible. Initial concentration of 0.5 × 10⁴cells/mL was used. The duration of this dose-response test was 72 hours. Following a range-finding test with nominal test item concentrations of 0.00100, 0.0100, 0.100, 1.00 and 10.0 mg/L, solvent control and control, a main test with nominal concentrations of 1.08, 3.36, 10.4, 32.3, 100 mg/L, solvent control and control was performed.  Six replicates were performed for the control as well as for the solvent control and three for each test item concentration. The test was performed in 100 mL Erlenmeyer flasks under continuous illumination at 71.2 – 85.3 µEm^-2s^-1at cell culture level. The final volume in each test vessel was 50 mL. Analytical samples were taken and analysed in the main test at 0 hours (initial value) from fresh test solutions and at 72 hours from aged test solutions from all test concentrations, solvent control and control. The EC_10,20,50were determined by probit analysis. The NOEC and LOEC were determined using pair-wise multiple comparison tests.

The EC50-value for growth rate (ErC50) was 60.0mg/L (nominal). The EC50-value for yield (EyC50) was11.5mg/L (nominal). For growth rate the NOEC was determined to be at 10.4 mg/L (nominal) and the LOEC at 32.3 mg/L. For yield the NOEC was determined to be at 3.36 mg/L and the LOEC at 10.4 mg/L (nominal).

Description of key information

In the acute algae study, the EC50-value for growth rate (ErC50) was 60.0 mg/L (nominal). The EC50-value for yield (EyC50) was11.5mg/L (nominal). For growth rate the NOEC was determined to be at 10.4 mg/L (nominal) and the LOEC at 32.3 mg/L. For yield, the NOEC was determined to be at 3.36 mg/L and the LOEC at 10.4 mg/L (nominal).

Key value for chemical safety assessment

EC50 for freshwater algae:

60 mg/L