4,5,6,7-tetrahydro-1H-benzotriazole

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:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study without restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4,5,6,7-tetrahydro-1H-benzotriazole

EC Number:

229-858-6

EC Name:

4,5,6,7-tetrahydro-1H-benzotriazole

Cas Number:

6789-99-7

Molecular formula:

C6H9N3

IUPAC Name:

4,5,6,7-tetrahydro-1H-1,2,3-benzotriazole

Details on test material:

Physical state, appearance: solid, white
Storage conditions: room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

samples of each test item concentration and the control collected at exposure initiation and at exposure termination were analyzed. Each sample in a volume 2 mL (i.e. control sample, test sample, sample fortified with the standard) was collected and diluted with deionized water (if necessary) and applied to the chromatographic column in a volume of 10 μL.
Given the description above, some sample was diluted before chromatographic analysis. This was done to ensure the result fits within the range of the respective standard curve.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: AAP medium (US EPA), recommended in OECD 201
The test item in the amount of 1000.3 mg was weighed in a glass crystallizer, quantitatively transferred to a volumetric flask by multiple washing with the AAP medium, and filled up 1 L [SOP/W/7]. The content of flask was heterogeneous (visible non-dissolved particles), therefore it was sonicated for
10 minutes [SOP/W/78]. The resulting test item concentration of 1000 mg/L was visually homogeneous and transparent. The lower test item concentrations were prepared by sequential dilutions with the AAP medium in a ratio 1 : 1, volume per volume [9] (Table 4). The control was AAP
medium (800 mL). Additionally, one replicate of each treatment was used as a background (i.e. an extra replicate without the algae used for the purpose of spectrophotometric measurements) and another one replicate of each treatment was prepared for chemical determinations at exposure initiation (replicate with the algae)

The alkalinity determined for AAP medium was 13.6 mg/L as NaHCO3, the hardness was 18.9 mg/L as CaCO3

- Evidence of undissolved material (e.g. precipitate, surface film, etc):
The test item concentrations were visually homogeneous and transparent directly after preparation and throughout exposure period.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: SAG 61.81
- Source (laboratory, culture collection): Algae Collection University of Göttingen, Germany, cultivated at the performing institute.
- Method of cultivation: Algae were transferred from agar bevels to the fresh medium (for Pseudokirchneriella subcapitata AAP medium is recommended, Appendix 2) in 250 mL Erlenmeyer flask and incubated in 21 – 24 C in constant illumination (pre-culture). The algae culture was renewed (inoculated to a new medium) twice a week in sterile conditions.
The cell density in the four-day-old algal preculture was determined by counting the number of cells in the Bürker chamber under a microscope. It was 3.950 x 106 cells/mL. The pre-culture was used to inoculate every replicates. The initial algae cells density was 1 x 104 cells per mL.The pre-culture was used for inoculation of the test concentrations and the control.

ACCLIMATION
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: no

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.1 – 22.4ºC

pH:

exposure initiation 7.33 – 7.54
exposure termination 7.37 – 8.71

Conductivity:

The conductivity of the test medium measured at exposure initiation in the control, before split up into replicates, was 84.9 μS/cm

Nominal and measured concentrations:

nominal: 1000, 500, 250, 125, 62.5 mg/L (with a separation factor of 2.0) plus the control

Details on test conditions:

TEST SYSTEM
- Test vessel: glass Erlenmeyer flasks 250 mL
- Type (delete if not applicable): closed with air permeable stoppers
- Material, size, headspace, fill volume: 100 mL
- Aeration: none
- Initial cells density: 10E+04 cells/mL

- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS

- Photoperiod: The tests were performed under constant illumination; fluorescent lighting was used (six 24-W light bulbs). In the preliminary test, the light intensity was measured at exposure initiation and at exposure termination [SOP/W/39]. In the definitive test, the light intensity was measured using lux meter with a 2 receptor at exposure initiation and every 24 h.
- Light intensity and quality: 6290 – 6450 lux
- transmittance of each test item concentration and the control: measured at 670 nm wavelength in an additional replicate without algae. The transmittance values were in the ranges of 97.9 – 99.3% at exposure initiation and 99.7 – 100.0% at exposure termination when compared with the control


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometric measurement
- Observations of the morphology of the algae were performed at exposure termination. In all test item concentrations no differences in shape, size and colour of algae cells were observed as compared to the algae cells in the control

TEST CONCENTRATIONS
- Range finding study: yes, 72h
The preliminary test was conducted using concentrations: 100, 10, 1.0 and 0.1 mg/L plus the control. The average transmittance values were in the range of 99.2 – 99.4% at exposure initiation and 99.8 – 100.0% at exposure termination when compared with the control. Hence, the indirect method was adequate to determine the number of algal cells.
The growth rate inhibition after 72 hours of exposure was 1.5% in the test item concentration of 0.1 mg/L, 0.7% in the test item concentration of 1.0 mg/L, 1.6% in the test item concentration of 10 mg/L and 2.2% in the test item concentration of 100 mg/L when compared to the control.
The yield inhibition after 72 hours of exposure was 6.8% in the test item concentration of 0.1 mg/L, 3.5% in the test item concentration of 1.0 mg/L, 7.6% in the test item concentration of 10 mg/L and 10.3% in the test item concentration of 100 mg/L when compared to the control.

Stability test:
In the stability test, the test item concentration of 100 mg/L and the control were chemically determined, using a validated liquid chromatographic method with DAD detection. The test item concentration of 100 mg/L was visually homogeneous and transparent. In sample collected at test initiation the determined test item concentration was 104.07 mg/L. At test termination the determined test item concentration was 106.22 mg/L. Therefore, the test item was stable under test conditions.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

588.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

195.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

253.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

106.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

At exposure initiation the determined test item concentrations were in the range of 98.04 – 100.03% of the nominal concentration. The results confirm that the test item concentrations were prepared correctly. At exposure termination the determined test item concentrations were in the range of 97.89 – 102.32% of the nominal concentration. Therefore, the test item concentrations were stable under test conditions.

GROWTH RATE:
Statistical tests based on the growth rate data were the Shapiro-Wilk’s Test on Normal Distribution which confirmed normal distribution of the data, the Levene’s Test on Variance Homogeneity (with Residuals) which showed that the variances were homogeneous, and the Williams Multiple Sequential t-test Procedure which showed a significant difference between the test item concentrations in the range of 125 – 1000 mg/L and the control and the control. Therefore, the LOEC/72 h value is 125 mg/L and the NOEC/72 h value is 62.5 mg/L.

YIELD:
Statistical tests based on the yield data were the Shapiro-Wilk’s Test on Normal Distribution which confirmed normal distribution of the data, the Levene’s Test on Variance Homogeneity (with Residuals) which showed that the variances were homogeneous, and the Williams Multiple Sequential t-test Procedure which showed a significant difference between the test item concentrations in the range of 125 – 1000 mg/L and the control. Therefore, the LOEC/72 h value is 125 mg/L and the NOEC/72 h value is 62.5 mg/L.


In the definitive test, the following validity criteria specified in OECD Guideline No. 201 (2006) were met:
- the biomass in the control increased by a factor of 75.2 within the 72-hour test period (criterion: at least a 16-fold growth),
- the coefficient of variation of the mean specific growth rate after the 72-hour test period (exposure initiation – exposure termination) in the control culture was 0.7% (criterion: it must not exceed 7%).
- the mean coefficient of variation for the section-by-section growth rate in the control culture was 7.2% (criterion: it must not exceed 35%).

Results with reference substance (positive control):

conducted: June 23, 2017 and June 26, 2017.
Six concentrations of the reference material ranging from 0.56 to 10 mg/L were used. There were three replicates of each concentration of reference material and six replicates of the control.
The results are within the range given in the literature reference.
ErC50 72h 2.57mg/L
ErC10 72h 1.77mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes