Methyl 1-methyl-4-[(methylphenylhydrazono)methyl]pyridinium sulphate

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:

23Jan2019 - 10May2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 1-methyl-4-[(methylphenylhydrazono)methyl]pyridinium sulphate

EC Number:

269-503-2

EC Name:

Methyl 1-methyl-4-[(methylphenylhydrazono)methyl]pyridinium sulphate

Cas Number:

68259-00-7

Molecular formula:

C14H16N3.CH3O4S

IUPAC Name:

methyl 1-methyl-4-[(methylphenylhydrazono)methyl]pyridinium sulphate

Test material form:

solid: particulate/powder

Details on test material:

Batch 1065826

Specific details on test material used for the study:

Identification: Methyl 1-methyl-4-[(methylphenylhydrazono)methyl]pyridinium sulphate
Batch (Lot) Number: 1065826
Expiry date: 13 November 2021 (expiry date)
Physical Description: Yellow powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, a sample was taken from the stock solution at the start of the test for possible analysis. The method of analysis is described in the appended Analytical Report.

Frequency at t=0 h and t=72 h.
Volume 3.0 mL from the approximate centre of the test vessels.
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at 0.10 mg/L but without algae and samples for analysis were taken at the start and at the end of the test period.

Additionally, reserve samples of 3.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

Test samples were stored in the freezer (≤ -15°C). On the day of analysis, the test samples were thawed at room temperature. Afterwards, the samples were diluted in a 3:1 (v:v) ratio with 0.3% formic acid in acetonitrile and analyzed.

If necessary, the samples were further diluted with 25/75 (v/v) acetonitrile/0.1% formic acid in test-medium to obtain concentrations within the calibration range.

Test organisms

Test organisms (species):

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

Details on test organisms:

Species: Raphidocelis subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.

Reason for selection This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Freshwater Algae Culture
Stock culture Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Study design

Test type:

static

Water media type:

other: M2; according to the OECD 201 Guideline

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

21-24°C, constant within ±1°C

pH:

7.9-8.2

Nominal and measured concentrations:

Nominal concentration, Geometric mean of exposure concentration
0.010, 0.0060
0.032, 0.025
0.10, 0.085
0.32, 0.31
1.0, 1.0

Control without Algae:
Nominal concentration, Geometric mean of exposure concentration
0.10, 0.080

Details on test conditions:

Test duration: 72 hours
Test type: Static
Test vessels: 250 mL, all-plastic culture flasks, containing 30 mL of test solution
Test Medium: M2
Cell density: An initial cell density of 1 x 104 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 73 to 77 μE.m-2.s-1.
Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

ERC50

Effect conc.:

>= 0.28 - <= 0.31 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

EYC50

Effect conc.:

>= 0.078 - <= 0.09 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield inhibition

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.025 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.025 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield inhibition

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata, the test item inhibited growth rate and yield of this freshwater algae species significantly at average exposure concentrations of 0.085 mg/L and higher.
The 72h-EC50 for growth rate inhibition (ERC50) was 0.29 mg/L with a 95% confidence interval ranging from 0.28 to 0.31 mg/L.
The 72h-EC50 for yield inhibition (EYC50) was 0.084 mg/L with a 95% confidence interval ranging from 0.078 to 0.090 mg/L.
The 72h-NOEC for growth rate inhibition was 0.025 mg/L based on statistical significance.
The 72h-NOEC for yield inhibition was 0.025 mg/L based on statistical significance.

Executive summary:

The objective of the study was to evaluate the test item for its ability to generate toxic effects in Raphidocelis subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10, EC20 and EC50 for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011.

The batch of the test item was a yellow powder with a purity of 99.9% and completely soluble in test medium at the concentrations tested.

A final test was performed based on the results of a preceding combined limit/range-finding test. Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L. The initial algal cell density was 104 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of exposure.

The measured concentration of the stock solution was 111% of nominal, indicating appropriate preparation of the stock solution. The measured test concentrations at the start of the test were generally at the level of nominal, i.e. were 79 – 105% of nominal. During the exposure period, the concentrations decreased to 57 – 94% of initial at the end of the test. Based on these results, the average exposure concentrations were calculated and used to express effect parameters.

Inhibition of growth rates and yield increased with increasing concentration of the test item from 0.085 mg/L upwards resulting in 74 and 99% inhibition, respectively, at the highest test concentration. Statistically significant inhibition of growth rates and yield was found at test concentrations of 0.085 mg/L and higher.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters obtained in this study are summarized below:

The 72h-EC50 for growth rate inhibition (ERC50) was 0.29 mg/L with a 95% confidence interval ranging from 0.28 to 0.31 mg/L.

The 72h-EC50 for yield inhibition (EYC50) was 0.084 mg/L with a 95% confidence interval ranging from 0.078 to 0.090 mg/L.

The 72h-NOEC for growth rate inhibition was 0.025 mg/L based on statistical significance.

The 72h-NOEC for yield inhibition was 0.025 mg/L based on statistical significance.