Bis[3-[[4-[benzylmethylamino]phenyl]azo]-1,4-dimethyl-1H-1,2,4-triazolium] tetrachlorozincate(2-)

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 12 to 19, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

adopted 23th March 2006

Deviations:

yes

Remarks:

Light homogeneity: not in the 15% range. Temperature ranged between 22.7 and 25.1°C. These deviations are not expected to have a significant impact on the outcome of the study. The spacing factor of the concentrations was not kept constant

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations of the non-GLP preliminary tests: 1, 10, 100 mg/l nominal
- Concentrations of the definitive test: 400, 100, 25.0, 6.25, 1.56, 0.391 mg/l nominal concentration, corresponding to 350, 87.5, 21.9, 5.47, 1.37, 0.342 mg/l of the active ingredient.
Due to the very flat dose-response relationship, a spacing factor of 4 was used. However, due to a dilution error during preparation, the 100 mg/l concentration was prepared as a 50 mg/l test solution, what could be confirmed by the analytical determination. The spacing factor was consequently not kept constant.

Details on test solutions:

TEST VASSELS
400 ml beakers, all-glass, with 200 ml of test medium. The beakers were covered with black paper up until the 200 ml mark to ensure that illumination comes only from above and not from the sides.
The following test vessels were set up:
• Test solution (Tn); containing test medium, test item and duckweed (three replicates per concentration)
• Blank control (Bn); containing test medium and duckweed (six replicates)
Since the test item is soluble, the test solutions were prepared by respective dilutions of a stock solution (test item dissolved in STEINBERG medium) with STEINBERG medium prepared with ultra-pure water. The resulting solutions were used as the test solutions in the toxicity test.
Pure STEINBERG medium served as blank controls.
The pH of the STEINBERG medium was adjusted before the test to pH 5.5 ± 0.2.
Young and rapidly growing plants without visible lesions or discoloration (chlorosis) from an exponentially growing monoculture of Lemna minor were distributed into the test vessels (9-12 fronds per replicate, 2-4 fronds per plant).
The vessels were randomly placed in the incubation chamber at the start of the test and randomly repositioned after the observations made at days 3 and 5 (Beakers were incubated on a black non-reflecting surface).

Test organisms (species):

Lemna minor

Details on test organisms:

- Test organism:
Exponential growing plant monoculture of Lemna minor (Umweltbundesamt, FGIII 2.5, Überwachungsverfahren Abwasserentsorgung, Schichauweg 58, D-12307 Berlin)

- Pre-culture:
Exponentially growing plant monoculture of Lemna minor (the culture was visibly free from contamination by other organisms such as algae and protozoa).

- Culture:
All-glass vessel containing sterile STEINBERG medium (Table 2) and the exponentially growing plant monoculture. Healthy Lemna minor plants consist of colonies comprising 2-5 fronds.

- Cultivation:
At least seven days before testing, sufficient colonies were transferred aseptically into fresh sterile medium and cultured for 7-10 days under the conditions of the test.

- Illumination:
Intensity: Continuous (6500–10000 lux) from Osram Fluora L18W77 (Osram AG, Winterthur, Switzerland) and CH Lighting F18T8/6500K EUP (CH Lighting CO., Ltd., China) Homogeneity: ±15%

- Temperature: 24 ± 2 °C

- Control of sensitivity:
Yearly, with 3,5-dichlorophenol

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

Measured in an additional glass vessel containing STEINBERG medium and Lemna, at least daily. The temperature ranged between 22.7 and 25.1°C, which is slightly out of the required range of 24±2°C.

pH:

5.5 at the beginning of the test; the pH value in the blank control should not increase by more than 1.5 units during the test
Determined in the combined replicate test solutions at the beginning and at the end of the test. The pH value in the control drifted by 1.2 units during the whole test period, which is therefore in the range allowed by the guideline (required: not more than 1.5).

Nominal and measured concentrations:

DOC analyses of the test concentrations were conducted at the beginning of the test, as well as after 3, 5 and 7 days of exposure

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

17.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.977 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

The test item did not show any signs of precipitation, at any of the test concentrations.

Non-GLP preliminary tests for Lemna toxicity

Prior to the definitive test, a non-GLP range finding test with the nominal concentrations of 1, 10 and 100 mg/l of Basic Red 46 (test item as a whole) was performed. Since the test item is soluble, the test solutions were prepared by dilution of a stock solution.

The percentage of growth inhibition for the different concentrations in this non-GLP range finding test are shown below:

Nominal concentration of T.M. (mg/l)	% inh. Growth rate of frond number	% inh. yield of frond number
0	0	0
1	11	24
10	20	39
100	38	63

Test concentrations

The test concentrations of test item during the 7 day test period were determined by DOC analysis at the beginning of the test, as well as after 3, 5 and 7 days of exposure.

The stability of the test concentrations was assessed by considering all concentrations together: since the two higher concentrations showed a good stability (89-103 % of the nominal) it was assumed that the relative variability of the two middle concentrations (76-125%, with two outliers; 2 % and 27 %) was rather due to measurement variability rather than to true concentration variability (this assumption is also supported by the fact that no precipitations were observed).

For the two lowest concentrations, which are at or below the LOQ of 0.5 mg/l, the "measured" concentrations have been replaced by the respective nominal concentration multiplied by the average recovery rate of the 6.25, 25, 50 and 400 mg/l concentration, for each day.

All in all, because the DOC analytics did not allow to determine the exact test concentrations but nonetheless indicated that they remained rather constant over the whole 7-day test period and that they corresponded to the nominal concentrations, the effective concentrations (E_rC₅₀and E_yC₅₀) were assessed based on the nominal concentrations of the active ingredient.

Effects on frond number

With respect to growth rate inhibition, the following effects as compared to the untreated controls were observed: 97 % at 400 mg/l, 69 % at 50.0 mg/l, 53 % at 25.0 mg/l, 37 % at 6.25 mg/l, 9 % at 1.56 mg/l and 10 % at 0.391 mg/l.

With respect to yield inhibition, the following effects as compared to the untreated controls were observed: 99 % at 400 mg/l, 87 at 50.0 mg/l, 77 % at 25.0 mg/l, 61 % at 6.25 mg/l, 20 % at 1.56 mg/l and 22 % at 0.391 mg/l.

The results of the toxicity assessment of test item to the duckweed Lemna minor, for the endpoint frond number, are summarized in the following table showing E_rC_xand NOE_rC values (Growth Rate) and E_yC_xand NOE_yC values (Yield) based on the nominal concentrations of the active ingredient:

Nominal concentration of A.I (mg/l)	ErC10	ErC50	NOErC	EyC10	EyC50	NOEyC
Estimate	0.977	17.1	<0.391	0.276	4.62	<0.391
Lower 95 %-cl	0.504	13.6		0.105	3.3
Upper 95 %-cl	1.57	21.5		0.517	6.37

Effects on dry weight

In the data of the dry weight inhibition each of the 4 lower test concentration showed one replicate whose inhibition was dramatically different from the 2 other replicates. Since this would result in an inflated variance, and consequently a poor statistical fit and very wide confidence intervals, these 4 individual replicates were considered as outliers. The E_rC₅₀values computed with both datasets did not differ significantly (all original data: 5.68 mg/l; data without outliers: 6.88 mg/l), but the reliability of the confidence intervals was increased (all original data: 2.83–10.7 mg/l; data without outliers: 5.40–8.64 mg/l).

With respect to growth rate inhibition, the following effects as compared to the untreated controls were observed: 105 % at 400 mg/l, 103 % at 50.0 mg/l, 84 % at 25.0 mg/l, 45 % at 6.25 mg/l, 15 % at 1.56 mg/l and 7 % at 0.391 mg/l.

With respect to yield inhibition, the following effects as compared to the untreated controls were observed: 100 % at 400 and 50.0 mg/l, 94 % at 25.0 mg/l, 70 % at 6.25 mg/l, 30 % at 1.56 mg/l and 16 % at 0.391 mg/l.

The results of the toxicity assessment of test item to the duckweed Lemna minor, for the endpoint dry weight, are summarized in the following table showing E_rC_xand NOE_rC values (Growth Rate) and E_yC_xand NOE_yC values (Yield) based on the nominal concentrations of the active ingredient:

Nominal concentration of A.I (mg/l)	ErC10	ErC50	NOErC	EyC10	EyC50	NOEyC
Estimate	1.118	6.88	1.56	0.389	2.87	0.391
Lower 95 %-cl	0.601	5.4		0.249	2.4
Upper 95 %-cl	1.69	8.64		0.543	3.42

 Appearance of the plants at the end of the test

In the blank control the plants were healthy, with roots reaching the bottom of the test vessel.

At 0.391 and 1.37 mg/l, the plants looked like the blanks’, except for the length of the roots, which was only about half of the blanks’.

At 6.25 mg/l, while the roots reached the bottom of the test vessel, the plants were smaller than the blanks’ and their leaves were less coloured and narrower.

At 25.0 mg/l, the plants were similar to those at 6.25 mg/l, but the leaves showed some signs of chlorosis and were in general very small.

At 100 mg/l, even though the roots reached the bottom, the leaves were very small with great signs of chlorosis.

At 400 mg/l, the roots were only 2/3 of the blanks’ and all leaves were coloured by the dye, suggesting a very important lack of photosynthetic activity.

 Evaluation of the test results

Calculation of average growth rate inhibition

The average specific growth rate (µ) in each test vessel is calculated as the logarithmic increase of the growth variables (fronds numbers and one other measurement variable e.g. total frond area, dry weight or fresh weight).

The percentage growth rate inhibition in each sample (I_r) is calculated as the difference between the mean growth rate of the controls (µ_C) and the growth rate at each test item concentration (µ_T)

Calculation of yield inhibition

The yield is calculated as the value of the measurement variable at the end of the test minus the value of the measurement variable at the start, for each single vessel of controls and treatments.

For dry weight, the starting biomass is determined on the basis of a sample of fronds taken from the same batch used to inoculate the test vessels.

Definition and determination of the effect concentrations (EC_x)

The effective concentration E_rC_xwith respect to growth rate, and E_yC_xwith respect to yield, is the concentration of test item which causes an x % reduction in growth rate or yield, respectively. These values can be determined for any of the variables considered (frond number and one other measurement variable e.g. total frond area, dry weight or fresh weight).

The effect concentrations E_rC_xand E_yC_xwre determined with ToxRat® Standard Version 2.10 (ToxRat® Solutions GmbH, Alsdorf, Germany). For the calculation of the EC_xand associated confidence intervals, inhibitions below 0 % and inhibitions above 100 % were set to 0.1 % and 100 %, respectively.

Definition and determination of the no observed effect concentrations (NOE_rC and NOE_yC)

The no observed effect concentration (NOE_rC or NOE_yC) is the highest concentration tested in which the test item is observed to have no statistically significant reducing effect on growth when compared with the control.

The statistical significance of differences between effects observed for control and test item treatments were determined by Dunnett’s test using the ToxRat® Standard Version 2.10 (ToxRat® Solutions GmbH, Alsdorf, Germany).

Validity criteria fulfilled:

yes

Remarks:

The doubling time of frond number in the control must be less than 2.5 days (60 h), corresponding to approximately a seven-fold increase in seven days and an average specific growth rate of 0.275 d-1.

Conclusions:

ErC50 (7d, nominal a.i.) = 17.1 mg/l.

Executive summary:

Method

The inhibitory effects of test item to the duckweed Lemna minor were investigated over a period of 7 days, based on the frond number and biomass (dry weight), following the guideline OECD 221.

The test item is solid, well soluble (34.3 g/l in water) and has a high purity. The test solutions were prepared by respective dilutions of a stock solutionin STEINBERG medium.

The test was performed at of 400, 50.0, 25.0, 6.25, 1.56, 0.391 mg/l nominal concentrations of test item, corresponding to 350, 43.7, 21.9, 5.47, 1.37, 0.342 mg/l of the active ingredient.

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

The test concentrations of test item during the 7 day test period were determined by dissolved organic carbon (DOC) analysis at the beginning of the test, as well as after 3, 5 and 7 days of exposure. These analyses confirmed that the test item was fully dissolved, and that the concentrations were kept rather constant over the whole 7-day test period. Therefore, the effective concentrations (E_rC₅₀and E_yC₅₀) were assessed based on the nominal concentrations of the active ingredient.

Observation

The two endpoints frond number and biomass (dry weight) were investigated at days 3, 5 and 7, and each of them were assessed as growth rate and yield.

The results of the toxicity assessment of the substance to the duckweed Lemna minor are summarized in the following table showing EC_xvalues based on the geometric mean of the measured concentrations:

Nominal concentration of A.I (mg/l)	EC50	EC10	NOEC
Frond Number, Growth Rate	17.1	0.98	<0.391
Frond Number, Yield	4.62	0.28	<0.391
Dry Weight, Growth Rate	6.88	1.12	1.56
Dry Weight, Yield	2.87	0.39	0.391

Conclusion

The 7 day E_rC₅₀of test substance on the duckweed Lemna minor is 17.1 mg/l. This value is based on the nominal concentrations of the active ingredient.

Description of key information

 ErC50 (7d, nominal a.i.) = 17.1 mg/l.

Key value for chemical safety assessment

EC50 for freshwater plants:

17.1 mg/L

EC10 or NOEC for freshwater plants:

0.977 mg/L

Additional information

Method

The inhibitory effects of test item to the duckweed Lemna minor were investigated over a period of 7 days, based on the frond number and biomass (dry weight), following the guideline OECD 221.

The test was performed at of 400, 50.0, 25.0, 6.25, 1.56, 0.391 mg/l nominal concentrations of test item, corresponding to 350, 43.7, 21.9, 5.47, 1.37, 0.342 mg/l of the active ingredient.

The test concentrations of test item during the 7 day test period were determined by dissolved organic carbon (DOC) analysis at the beginning of the test, as well as after 3, 5 and 7 days of exposure.

After 7 days of exposure the following results are available:

Nominal concentration of A.I (mg/l)	EC50	EC10	NOEC
Frond Number, Growth Rate	17.1	0.98	<0.391
Frond Number, Yield	4.62	0.28	<0.391
Dry Weight, Growth Rate	6.88	1.12	1.56
Dry Weight, Yield	2.87	0.39	0.391