3,6-bis(diethylamino)-9-[2-(methoxycarbonyl)phenyl]xanthylium tetrachlorozincate

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:

20 February 2017 to 05 April 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:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples for possible analysis were taken from all test concentrations and the control. Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period. Additionally, reserve samples of 1.4 mL were taken from all test solutions for possible analysis.
- Sampling method: At t = 0, t = 24 and t = 72 h, 1.4 mL was sampled. At the end of the exposure period, the replicates were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15 °C) until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The batch tested was completely soluble in test medium at the concentrations tested. Preparation of test solutions started with the highest concentration of 100 mg/L (combined limit/range-finding test) or 1.0 mg/L (full test) applying 15 to 17 minutes of magnetic stirring to accelerate dissolution of the test material in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The colour of the final test solutions in the combined limit/range-finding test ranged from light pink to dark red/violet with increasing test material concentrations. The colour of the test solutions in the final test ranged from colourless to light pink with increasing test material concentrations.
After preparation, volumes of 30 mL were added to each replicate of the respective test concentration. Subsequently, 0.6 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): Cultures were maintained for 4 days prior to the test

ACCLIMATION
- Culturing media and conditions (same as test or not): 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 to 24 °C. Light intensity was 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm. Stock culture medium was M1. Four days before the start of the test, cells from the algal stock culture were inoculated in pre-culture medium M2 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.
- Any deformed or abnormal cells observed: Not specified

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

22 to 24 °C

pH:

7.5 to 7.8

Nominal and measured concentrations:

- Nominal concentrations: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L
- Time weighted average concentrations: 33, 44 and 67 µh/L for the 0.10, 0.32 and 1.0 mg/L concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL
- Type: Capped
- Material, size, headspace, fill volume: Plastic cell culture vessels (Greiner), containing 30 mL of test solution
- Aeration: No, however algal cells were kept in suspension by continuous shaking
- Initial cell density: 1 x 10^4 cells per mL
- Control end cell density: 1.85 x 10^6
- No. of vessels per concentration (replicates): 3; 1 or 2 replicates of each test concentration without algae and 1 or 2 extra replicates of each test group for sampling after 24 hours.
- No. of vessels per control (replicates): 5 replicates (5 instead of 6 replicates of the control treatment were available for analysis because one of replicates fell from the shaking table and possibly part of the solution was lost; sufficient number of replicates was present to perform statistical analysis with sufficient power)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2 according to the OECD 201 guideline, formulated using Milli-RO water and with the following composition: NH4Cl 15 mg/L, MgCl2.6H2O 12 mg/L, CaCl2.2H2O 18 mg/L, MgSO4.7H2O 15 mg/L, KH2PO4 1.6 mg/L, FeCl3.6H2O 64 μg/L, Na2EDTA.2H2O 100 μg/L, H3BO3 185 μg/L, MnCl2.4H2O 415 μg/L, ZnCl2 3 μg/L, CoCl2.6H2O 1.5 μg/L,CuCl2.2H2O 0.01 μg/L, Na2MoO4.2H2O 7 μg/L and NaHCO3 50 mg/L.
- Ca/mg ratio: Hardness (Ca + Mg) 0.24 mmol/L (24 mg CaCO3/L)
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test. Temperature was continuously monitored in a temperature control vessel.

OTHER TEST CONDITIONS
- Photoperiod: Continuous
- Light intensity and quality: TLD-lamps with a light intensity within the range of 101 to 122 μE.m^-2.s^-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length =10 mm). Algal medium was used as blank.
- Other: At the end of the test microscopic observations were performed on the 0.32 mg/L concentration and the control to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: Yes
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes. The expected EC50 for growth rate inhibition was between 0.10 and 1.0 mg/L. The expected EC50 for yield inhibition was below 0.10 mg/L.

DATA HANDLING
- Calibration curve
Quantification of cell densities was based on a calibration curve. Cell density was plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of an algal suspension with different cell densities. The calibration curve was composed using linear regression. The software automatically calculates the cell densities based on this curve for the spectrophotometric measurements at the various points in time during the test period.

- Comparison of average growth rates
The average specific growth rate for a specific period is calculated as the logarithmic increase in the biomass from the equation for each single vessel of controls and treatments:
µi-j = (lnXj - lnXi) / (tj - ti) (day^-1)
where:
μi-j = the average specific growth rate from time i to j
Xi = the biomass at time i
Xj = the biomass at time j

The average growth rate at each test material concentration is then compared with the control value and the percentage inhibition in growth rate is calculated:
%Ir = [(µC - µT) / µC] x 100
where:
%Ir = percent inhibition in average specific growth rate
μC = mean value for average specific growth rate in the control group
μT = average specific growth rate for the treatment replicate

-Yield
The percent inhibition in yield is calculated for each treatment replicate as follows:
%Iy = [(YC - YT) / YC] x 100
where:
%Iy = percent inhibition of yield
YC = mean value for yield in the control group
YT = value for yield for the treatment replicate

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

41 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

37 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

Table 1 shows group mean growth rates and the percentages of growth rate inhibition (total test period) whereas Table 2 shows the values at different time intervals. The group mean yields and the percentages of yield inhibition are summarized in Table 3.
Growth rates were in the range of the controls at the three lowest concentrations during the 72-hour test period, whereas the growth rate of algae exposed to the two highest concentrations were increasingly reduced.
Yield was stimulated rather than inhibited at the two lowest concentrations tested. Inhibition of yield progressed from 6.9 % at the TWA concentration of 33 µg/L and reached 99 % at 44 µg/L.
Statistically significant inhibition of growth rate and yield was found at the two highest concentrations tested.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 44 µg/L when compared to the control.

ACCEPTABILITY OF THE TEST
1. In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 185).
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 10 %).
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 % (i.e. 2.2 %).

Results with reference substance (positive control):

The reference test was carried out to check the sensitivity of the test system as used by the testing facility. Algae were exposed for 72 hours to K2Cr2O7 concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L and to a control. The initial cell density was 1.0 x 10^4.
Potassium Dichromate significantly inhibited the growth rate of this fresh water algal species at nominal concentrations of 0.56 mg/L and higher.
The EC50 for growth rate inhibition (72 h ERC50) was 1.2 mg/L with a 95 % confidence interval ranging from 1.1 to 1.2 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. The observed 72 h ERC50 for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (72 h EYC50) was 0.43 mg/L with a 95 % confidence interval ranging from 0.42 to 0.44 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L. The observed 72 h EYC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure α = 0.05, one-sided, smaller).
Computation of ECx values were done only with the three highest groups for which measureable concentrations were available. Calculation of ECx values was based 3-parameters cumulative distribution function (CDF; non-linear regression).
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Table 1: Percentage inhibition of growth rate (total test period) during the final test

Nominal concentration (mg/L)	Group Mean Growth Rate	Std. Dev.	n	% Inhibition
Control	1.738	0.0379	5	-
0.010	1.768	0.0702	3	-1.7
0.032	1.744	0.0164	3	-0.3
0.10 (33)	1.716	0.0128	3	1.3
0.32 (44)	0.394*	0.1270	3	77.3
1.0 (67)	0.063*	0.1086	3	96.4

( ) Between brackets TWA concentrations are given (µg/L)

*Effect statistically significant

 

Table 2: Percentage inhibition of growth rate at different time intervals during the final test

Nominal concentration (mg/L)	n	0 to 24 h		24 to 48 h		48 to 72 h
		Mean	% Inhibition	Mean	% Inhibition	Mean	% Inhibition
Control	5	1.577	-	1.919	-	1.718	-
0.010	3	1.743	-10.5	1.802	6.1	1.757	-2.3
0.032	3	1.777	-12.7	1.691	11.9	1.765	-2.7
0.10 (33)	3	1.666	-5.6	1.730	9.8	1.752	-2.0
0.32 (44)	3	0.538	65.9	0.598	68.8	0.045	97.4
1.0 (67)	3	0.000	100.0	0.303	84.2	-0.115	106.7

( ) Between brackets TWA concentrations are given (µg/L)

 

Table 3: Yield and percentage inhibition during the final test

Nominal concentration (mg/L)	Group Mean Yields	Std. Dev.	n	% Decrease
Control	183.9	20.63	5	-
0.010	202.9	42.21	3	-10.3
0.032	186.5	9.14	3	-1.4
0.10 (33)	171.2	6.57	3	6.9
0.32 (44)	2.4*	1.22	3	98.7
1.0 (67)	0.3*	0.44	3	99.9

( ) Between brackets TWA concentrations are given (µg/L)

*Effect statistically significant

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the test material reduced growth rate and inhibited the yield significantly at a TWA concentration of 33 µg/L and higher. The EC50 for growth rate inhibition (72 h-ERC50) was 41 µg/L with a 95 % confidence interval ranging from 35 to 48 µg/L. The EC50 for yield inhibition (72 h-EYC50) was 37 µg/L with a 95 % confidence interval ranging from 34 to 40 µg/L. The 72 h-NOEC for both growth rate and yield inhibition was 33 µg/L.

Executive summary:

The potential for the test material to cause acute toxicity to Pseudokirchneriella subcapitata was investigated in accordance with the standardised guidelines OECD 201 and EU Method C.3 under GLP conditions.

The batch tested was completely soluble in test medium at the concentrations tested. A full test was performed based on the results of a preceding combined limit/range-finding test. Six 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 10^4 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

At the start of the test, the actual test concentrations in the three highest groups were in agreement with nominal, i.e. 94 to 106 % of nominal. After 24 hours of exposure the measured concentrations were below the limit of detection of the analytical method (i.e. below 0.048 mg/L). The measured concentrations in the two lowest groups were below the limit of detection already at the start of the test. Based on these results, the Time Weight Average (TWA) concentrations were 33, 44 and 67 µg/L at nominally 0.10, 0.32 and 1.0 mg/L, respectively.

Growth rates were in the range of the controls at the three lowest concentrations during the 72-hour test period, whereas the growth rate of algae exposed to the two highest concentrations were increasingly reduced. Yield was stimulated rather than inhibited at the two lowest concentrations tested. Inhibition of yield progressed from 6.9 % at the TWA concentration of 33 µg/L and reached 99 % at 44 µg/L. Statistically significant inhibition of growth rate and yield was found at the two highest concentrations tested.

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

Under the conditions of this study, the test material reduced growth rate and inhibited the yield significantly at a TWA concentration of 33 µg/L and higher. The EC50 for growth rate inhibition (72 h-ERC50) was 41 µg/L with a 95 % confidence interval ranging from 35 to 48 µg/L. The EC50 for yield inhibition (72 h-EYC50) was 37 µg/L with a 95 % confidence interval ranging from 34 to 40 µg/L. The 72 h-NOEC for both growth rate and yield inhibition was 33 µg/L.

Description of key information

Under the conditions of this study, the test material reduced growth rate and inhibited the yield significantly at a TWA concentration of 33 µg/L and higher. The EC50 for growth rate inhibition (72 h-ERC50) was 41 µg/L with a 95 % confidence interval ranging from 35 to 48 µg/L. The EC50 for yield inhibition (72 h-EYC50) was 37 µg/L with a 95 % confidence interval ranging from 34 to 40 µg/L. The 72 h-NOEC for both growth rate and yield inhibition was 33 µg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

41 µg/L

EC10 or NOEC for freshwater algae:

33 µg/L

Additional information

The potential for the test material to cause acute toxicity to Pseudokirchneriella subcapitata was investigated in accordance with the standardised guidelines OECD 201 and EU Method C.3 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The batch tested was completely soluble in test medium at the concentrations tested. A full test was performed based on the results of a preceding combined limit/range-finding test. Six 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 10^4 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

At the start of the test, the actual test concentrations in the three highest groups were in agreement with nominal, i.e. 94 to 106 % of nominal. After 24 hours of exposure the measured concentrations were below the limit of detection of the analytical method (i.e. below 0.048 mg/L). The measured concentrations in the two lowest groups were below the limit of detection already at the start of the test. Based on these results, the Time Weight Average (TWA) concentrations were 33, 44 and 67 µg/L at nominally 0.10, 0.32 and 1.0 mg/L, respectively.

Growth rates were in the range of the controls at the three lowest concentrations during the 72-hour test period, whereas the growth rate of algae exposed to the two highest concentrations were increasingly reduced. Yield was stimulated rather than inhibited at the two lowest concentrations tested. Inhibition of yield progressed from 6.9 % at the TWA concentration of 33 µg/L and reached 99 % at 44 µg/L. Statistically significant inhibition of growth rate and yield was found at the two highest concentrations tested.

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

Under the conditions of this study, the test material reduced growth rate and inhibited the yield significantly at a TWA concentration of 33 µg/L and higher. The EC50 for growth rate inhibition (72 h-ERC50) was 41 µg/L with a 95 % confidence interval ranging from 35 to 48 µg/L. The EC50 for yield inhibition (72 h-EYC50) was 37 µg/L with a 95 % confidence interval ranging from 34 to 40 µg/L. The 72 h-NOEC for both growth rate and yield inhibition was 33 µg/L.