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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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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:
4 April - 7 July 2017
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)
Version / remarks:
23 March 2006, Annex 5 corrected 28 July 2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
20 May 2008, amended 7 December 2015, for the purpose of its adaptation to technical progress, by Commission Regulation (EU) 2016/266
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The following test flasks were set up:
- Test solution (Tn); containing test medium, test item and algae (three replicates per concentration)
- Blank control (Bn); containing test medium and algae (six replicates)
- Since the test item is soluble, the test solutions were prepared by respective dilutions of a stock solution. A 316 mg/l stock solution was prepared by adding the test item to sterile-filtered Algal medium (pH adjusted at 8.0 ± 0.2) in sterile glassware and stirred for 20 minutes. The respective dilutions of this stock solution were then prepared with sterile-filtered Algal medium (pH adjusted at 8.0 ± 0.2) and sterile glassware. 100 ml of each test solution were added to the sterile
test flasks and inoculated with an exponentially growing preculture of algae.
Pure Algal medium, also sterile filtered into sterile test flasks and inoculated with the exponentially growing preculture of algae mentioned above, served as blank controls. For the incubation, the test flasks were then placed on an orbital shaker at a nominal shaking speed of about 130 revolutions per min.
- Evidence of undissolved material: Not relevant. Since the test item is soluble, the test solutions were prepared by respective dilutions of a stock solution.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Test organism: Axenic slope culture of Desmodesmus subspicatus 86.81SAG (Collection of Algal Cultures, Institute of Freshwater Ecology, University of Göttingen, D-37073 Göttingen, Germany
- Culture: 250 ml flask containing 100 ml of sterile OECD medium inoculated with cell material from an axenic slope culture
- Illumination: Continuous (2000–3000 lux) from Osram Fluora L18W77 and Osram Daywhite L18W840 (Osram AG, Winterthur, Switzerland)
- Temperature: 21 to 24°C, maintained at ± 2°C in a thermo-controlled room
- Control of sensitivity: twice per year, with potassium dichromate
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
not reported
Test temperature:
Min: 22.2; Max: 24.5; Mean: 23.6
pH:
7.0 - 8.1
Dissolved oxygen:
not reported
Salinity:
not reported
Conductivity:
not reported
Nominal and measured concentrations:
The concentrations of p-nitrobenzoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) in the test media were measured by HPLC analysis at the beginning and after 24, 48 h and 72 h of exposure. These analyses revealed that the concentrations were stable over the 72 h test period (101 to 106% of the initially measured concentration) and were within 80-120% of the nominal concentration. The initially measured concentration was 54.4 mg/l and the measured concentration after 72 h of exposure was 55.2 mg/l based on p-nitrobenzoic acid (52.8% nominal concentration of p-nitrobenzoic acid in the test item).
Therefore, the effective concentrations ErCx/EyCx were assessed based on nominal concentration of the test item.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml flasks, all-glass, with 100 ml of test medium, shaken (130 rpm), capped with air permeable stoppers
- Test medium: Recommended OECD medium
- Preculture: Exponentially growing culture of Desmodesmus subspicatus
- Illumination: Source: continuous from Osram Fluora L 18W77 and Osram Daywhite L 18W840 (Osram AG, Winterthur, Switzerland)
- Light intensity: the light intensity amounts to about 3000 lux which corresponds to about 40 µE•m-2•s-2
- Homogeneity: the light intensity is maintained within ±15% from the average light intensity over the incubation area. The test vessels are repositioned every day within the incubation area to minimize variation.
- Temperature: 21 to 24°C, maintained at ± 2°C in a thermo-controlled room
- pH: Initially adjusted to 8 ± 0.2; the pH of the control medium should not increase by more than 1.5 units during the test
- Test type: Static exposure conditions over a period of 72 h
- Starting cell density: 0.5–0.85 µg/ml with respect to dry weight corresponding to about 2–5•103 cells/ml (i.e. OD680 of about 0.005 units)

RANGE FINDING STUDY
Prior to the definitive test a non-GLP range finding test with nominal concentrations of 1, 10 and 100 mg/l of p-Nitrobenzoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) was performed. The test item concentrations were calculated based on measured p-Nitrobenzoic acid considering a content of 52.8%. The HPLC determinations indicate that at least the p-Nitrobenzoic acid concentrations are decreasing, as they did not remain in the recommended 80-120% range of the nominal concentration and/or of the initial concentration.

EFFECT PARAMETERS MEASURED
- Cell density: Cell concentrations were determined every 24 h using a spectrophotometer at 680nm wavelength (Shimadzu UV 1800, Shimadzu Schweiz GmbH, Römerstr. 3, CH-4153 Reinach). Aliquots of 5 ml were removed from each test flask under sterile conditions. Cell concentrations at time 0 h were only determined in the blank controls.
Reference substance (positive control):
not required
Remarks:
Control of sensitivity: None – the culture is re-purchased annually
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
19.8 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.:
> 316 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.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
10.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
140 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Remarks:
- As the 3rd validity criterion was not fulfilled in the 1st definitive test, it had to be repeated. Only the results of the 2nd definitive test are reported here.

- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: yes (growth rate and yield)
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

With respect to the endpoint growth rate, no significant effects were observed as compared to the blank controls.
Based on these effects and the nominal concentrations of the test item, the median effect concentration with respect to growth rate (ErC50/0–3 days) of P-Nitrobenzoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) to Desmodesmus subspicatus was estimated to be >316 mg/l.
The low effect concentration with respect to growth rate ErC10 was calculated to be 19.8 mg/l (95% confidence limits: 11.9–30.9 mg/l).
The no observed effect concentration (NOErC) with respect to growth rate was determined to be 10.0 mg/l.

With respect to the endpoint yield (biomass production), no significant effects were observed as compared to the blank controls.
Based on these effects and the nominal concentrations of the test item, the median effect concentration with respect to yield (EyC50/0–3 days) of P-Nitrobenzoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) to Desmodesmus subspicatus was calculated to be 140 mg/l (95% confidence limits: 91.9–244 mg/l).
The low effect concentration with respect to yield EyC10 was calculated to be 10.1 mg/l (95% confidence limits: 2.08–21.1 mg/l).
The no observed effect concentration (NOEyC) with respect to yield was determined to be 10.0 mg/l.
Reported statistics and error estimates:
Statistical analysis was performed with respect to the no observed effect concentrations. The statistical analysis was conducted with the software ToxRat® Standard Version 3.2.1.
Validity criteria fulfilled:
yes
Remarks:
In the control cultures: average specific growth rate of 1.39 (>0.92)/ coefficient of variation of average specific growth rates: 1.4% (<=7%)/ mean coefficient of variation for section-by-section specific growth rates 12.9% (<=35%).
Conclusions:
The acute toxicity of P-Nitrobenzoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) (CAS no. 7394-38-9) to Desmodesmus subspicatus was determined in a 72 hour static test according to OECD guideline 201. The median effect concentration with respect to growth rate (ErC50/0–3 days) of P-Nitrobenzoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) to Desmodesmus subspicatus was estimated to be >316 mg/l nominal concentration. The no observed effect concentration (NOErC) with respect to growth rate was determined to be 10.0 mg/l nominal concentration. The results of the test can be considered reliable without restriction.
Executive summary:

The growth inhibitory effects of P-Nitrobenzoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) (CAS no. 7394-38-9) to the green alga Desmodesmus subspicatus were investigated according to test guideline OECD 201, over a period of 72 h.

The test item P-Nitrobenzoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) is solid, >98% pure, and very soluble (>=782 g/l in water at pH 6 and at 23 °C). Consequently, the test solutions were prepared by respective dilutions of a stock solution.

The nominal concentrations were 316, 100, 31.6, 10.0, 3.16 and 1.00 mg/l.

Three parallel test vessels were used for each test concentration of the test item and six vessels for the blank controls.

The test item P-Nitrobenzoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) consists of two components: P-Nitrobenzoic acid and 2,2’,2’’-nitrilotriethanol. Only P-Nitrobenzoic acid was determined in the test media by HPLC analysis. It is assumed that 2,2’,2’’-nitrilotriethanol remains stable during the test to a similar extent than P-Nitrobenzoic acid. The HPLC analyses revealed that the test concentrations were correctly dosed and that at least P-Nitrobenzoic acid could be satisfactorily maintained over the 72-h test period. Therefore, the effective concentrations ErCx/EyCx were assessed based on the nominal concentrations.

With respect to the endpoint growth rate, following effects were observed as compared to the blank controls: 18% at 316 mg/l, 22% 100 mg/l and 5% at 31.6 mg/l. No significant effects were observed

at 10.0, 3.16 and 1.00 mg/l.

With respect to the endpoint yield (biomass production), following effects were observed as compared to the blank controls: 53% at 316 mg/l, 62% 100 mg/l and 19% at 31.6 mg/. No significant effects were observed at 10.0, 3.16 and 1.00 mg/l.

The results of the growth rate (r) and yield (y) inhibition of P-Nitrobenzoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) to the green alga Desmodesmus subspicatus are summarized in the following table showing EC values and 95% confidence limits (cl) based on the nominal concentrations:

 Parameter, expressed as Measured concentration (mg/l)

 ErC10

 ErC50

 NOErC (1)

 Estimate

 19.8

>316 

 10.0

 Lower 95%-cl

 11.9

 n.d.

 

 Upper 95%-cl

30.9

 n.d.

 

 Parameter, expressed as Measured concentration (mg/l)

 EyC10

 EyC50

 NOEyC (1)

 Estimate

 10.1

140

10.0

 Lower 95%-cl

2.08

 91.9

 

 Upper 95%-cl

21.1

244

 

cl: confidence limit

(1) : determined by Dunnett's Test

The 72 h ErC50 value of P-Nitrobenzoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) (CAS no. 7394-38-9) on the green alga Desmodesmus subspicatus was >316 mg/l. This value is based on the nominal concentrations. All validity criteria were fulfilled.

Description of key information

The acute toxicity of p-Nitrobenzoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) (CAS no. 7394-38-9) to Desmodesmus subspicatus was determined in a 72 hour static test according to OECD guideline 201. The median effect concentration with respect to growth rate (ErC50/0–3 days) of p-Nitrobenzoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) to Desmodesmus subspicatus was estimated to be >316 mg/l nominal concentration. The no observed effect concentration (NOErC) with respect to growth rate was determined to be 10.0 mg/l nominal concentration. The results of the test can be considered reliable without restriction.

Key value for chemical safety assessment

Additional information