3,6-bis(diethylamino)-9-[2-(ethoxycarbonyl)phenyl]xanthylium chloride

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:

24 March 2017 to 17 August 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:

2008

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
- Sampling method: At t = 0, t = 24 and t = 72 h. 1.2 mL was taken. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Not applicable, samples were analysed on the day of sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test material was completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition. Preparation of test solutions started with a concentration of 100 mg/L applying an overnight period of magnetic stirring to accelerate dissolution of the test material in medium. Lower test concentrations were prepared by subsequent dilutions of the concentration of 100 mg/L in test medium. All test solutions were clear and ranged from light pink to red at the end of the preparation procedure. 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):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum and method of cultivation: 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.

ACCLIMATION
- Culturing media and conditions (same as test or not): Yes, the pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21 to 24 °C. Light intensity: 120 µE/m^2/s ± 15 % when measured in the photosynthetically effective wavelength range of 400 to 700 nm. The stock culture medium was M1 formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following components in mg/L: NaNO3 500, K2HPO4.3H2O 52, MgSO4.7H2O 75, Na2CO3.10H2O 54, C6H8O7.H2O 6, NH4NO3 330, CaCl2.2H2O 35, C6H5FeO7.xH2O 6, H3BO3 2.9, MnCl2.4H2O 1.81, ZnCl2 0.11, CuSO4.5H2O 0.08 and (NH4)6Mo7O24.4H2O 0.018. Pre-culture and the test took place in M2 medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

23 °C

pH:

7.8 to 8.1

Nominal and measured concentrations:

- The nominal concentrations were: 0.010, 0.022, 0.046, 0.10, 0.22, 0.46 and 1.0 mg/L
- The measured TWA concentrations were: 0.097, 0.22, 0.47, 1.2, 4.5, 13 and 76 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL, plastic cell culture vessels (Greiner)
- Type: capped
- Material, size, headspace, fill volume: 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 (provided by adding 0.6 mL of algal suspension)
- Control end cell density: 3.784 x10^6 cells/mL
- No. of vessels per concentration (replicates): 3 replicates of each test concentration; 1 or 2 replicates of each test concentration without algae; 1 or 2 extra replicates of each test group for sampling after 24 hours
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, 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.

TEST MEDIUM / WATER PARAMETERS
- 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
- Adjustment of pH: No
- Photoperiod: Continuously illuminated
- Light intensity and quality: TLD-lamps with a light intensity within the range of 93 to 111 µE.m^-2.s^-1.

EFFECT PARAMETERS MEASURED:
- Appearance of the cells: At the end of the final test microscopic observations were performed in the nominal concentration of 0.22 mg/L to observe for any abnormal appearance of the algae.
- Recording of cell densities: 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 and the extra replicates as background for the treated solutions.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 2.1 to 2.2
- Range finding study: Yes
- Test concentrations: 0, 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes, the expected EC50 for both growth rate and yield inhibition was between 0.10 and 1.0 mg/L (nominal concentration).

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.:

3.3 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI: 3.0 to 3.7 µg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.8 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % CI: 2.4 to 3.2 µg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.6 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.47 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

FINAL TEST
- Measured test material concentrations: Samples taken from all groups were analysed freshly. The actual concentrations were at the level of 66 to 98 % of nominal at the start of the test. Measured concentrations decreased strongly during the exposure and were at the level of 0.0049-1.4 % of initially measured at the end of the test. Based on these results, the time weighted average (TWA) concentrations were calculated (Table 1).
The observed decrease of concentrations cannot be explained by an adsorption to algal biomass because strong decrease of actual concentration was also observed in the solution incubated without algae.

- Inhibition of growth rate and inhibition of yield
Results can be seen in Tables 2 and 3.
A very sharp increase of toxicity was observed at the TWA concentration of 4.5 µg/L for both growth rate and yield inhibition. Below this concentration the observed effects were low, whereas at 4.5 µg/L and above, nearly total inhibition was observed.
Statistically significant inhibition of growth rate was found at test concentrations of 4.5 µg/L and higher, whereas for yield was statistically significantly inhibited at 1.6 µg/L and higher.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 4.5 µg/L when compared to the control.

ACCEPTABILITY OF THE TEST
- In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 378).
- The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35 % (i.e. 18 %).
- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 % (i.e. 4.1 %).

Results with reference substance (positive control):

- Results with reference substance valid: Yes
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, 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Potassium dichromate inhibited the growth rate at nominal concentrations of 0.32 mg/L and higher.
The EC50 for growth rate inhibition (72 h ERC50) was 0.99 mg/L with a 95 % confidence interval ranging from 0.97 to 1.0 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 0.37 mg/L with a 95 % confidence interval ranging from 0.36 to 0.37 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L. Hence, the 72 h EYC50 for the algal culture tested was just below the expected range.

Reported statistics and error estimates:

Determination of the NOEC and calculation of the EC50
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 (Williams Multiple Sequential t-test Procedure, α = 0.05, one-sided, smaller) or inhibition of yield (Step-down Jonckheere-Terpstra Test Procedure, α = 0.05, one-sided, smaller).
Additionally, the EC10 and EC20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993. Calculation of ECx values was based on Weibull analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding TWA concentrations of the test material.
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Table 1: Measured test concentrations versus nominal concentration during the final test

Nominal concentration (mg/L)	Measured concentrations (µg/L)			TWA (µg/L)
	t = 0 h	t = 48 h	t = 72 h
0.010	6.57	0.00833	0.0937	0.097
0.022	16.3	0.0187	0.195	0.22
0.046	42.9	0.0347	0.282	0.47
0.10 (without algae)	86.2	0.223	0.259	1.6
0.10	91.4	0.141	0.00806	1.2
0.22	205	0.863	0.0213	4.5
0.46	441	3.35	0.0214	13
1.0	976	51.1	0.0689	76

Table 2: Growth rate and percentage inhibition for the total test period

TWA concentration (µg/L)	Mean	Std. Dev.	n	% Inhibition
Control	1.971	0.0818	6	-
0.97	2.010	0.0263	3	-2.0
0.22	1.993	0.0235	3	-1.1
0.47	1.967	0.0181	3	0.2
1.6	1.962	0.0233	3	0.5
4.5	0.180	0.1106	3	91*
13	0.027	0.0475	3	99*
76	0.090	0.1566	3	95*

*= Statistically significant effect

Table 3: Yield and percentage inhibition for the total test period

TWA concentration (µg/L)	Mean	Std. Dev.	n	% Inhibition
Control	377.4	78.40	6	-
0.97	416.2	33.59	3	-10.3
0.22	394.9	27.96	3	-4.6
0.47	364.4	19.66	3	3.4
1.6	359.3	25.54	3	4.8*
4.5	0.8	0.56	3	100*
13	0.1	0.16	3	100*
76	0.4	0.73	3	100*

*= Statistically significant effect

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the test material inhibited growth rate significantly at a TWA concentration of 4.5 µg/L and higher.
The EC50 for growth rate inhibition (72 h ERC50) was 3.3 µg/L with a 95 % confidence interval ranging from 3.0 to 3.7 µg/L. The EC50 for yield inhibition (72 h EYC50) was 2.8 µg/L with a 95 % confidence interval ranging from 2.4 to 3.2 µg/L. The 72 h NOEC values for growth rate and yield inhibition were 1.6 and 0.47 µg/L, respectively.

Executive summary:

The acute toxicity of the test material to algae was investigated in accordance with the standardised guidelines OECD 201 and EU Method C.3 under GLP conditions.

Pseudokirchneriella subcapitata was exposed to the test material for 72 hours under static conditions. The test material was 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 exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to 0.010, 0.022, 0.046, 0.10, 0.22, 0.46 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.

Samples taken from all groups were analysed freshly. The actual concentrations were at the level of 66 to 98 % of nominal at the start of the test but decreased to 0.0049 to 1.4 % of initially measured at the end of the test. Based on these results, the time weighted average (TWA) concentrations were calculated to be 0.97, 0.22, 0.47, 1.6, 4.5, 13 and 76 µg/L at nominal concentrations of 0.010, 0.022, 0.046, 0.10, 0.22, 0.46 and 1.0 mg/L, respectively.

A very sharp increase of toxicity was observed at the TWA concentration of 4.5 µg/L for both growth rate and yield inhibition. Below this concentration the observed effects were low, whereas at 4.5 µg/L and above, nearly total inhibition was observed. Statistically significant inhibition of growth rate was found at test concentrations of 4.5 µg/L and higher, whereas for yield was statistically significantly inhibited at 1.6 µg/L and higher. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 4.5 µg/L when compared to the control. The study met the acceptability and was considered valid.

Under the conditions of this study, the test material inhibited growth rate significantly at a TWA concentration of 4.5 µg/L and higher.

The EC50 for growth rate inhibition (72 h ERC50) was 3.3 µg/L with a 95 % confidence interval ranging from 3.0 to 3.7 µg/L. The EC50 for yield inhibition (72 h EYC50) was 2.8 µg/L with a 95 % confidence interval ranging from 2.4 to 3.2 µg/L. The 72 h NOEC values for growth rate and yield inhibition were 1.6 and 0.47 µg/L, respectively.

Description of key information

Under the conditions of this study, the test material inhibited growth rate significantly at a TWA concentration of 4.5 µg/L and higher.

The EC50 for growth rate inhibition (72 h ERC50) was 3.3 µg/L with a 95 % confidence interval ranging from 3.0 to 3.7 µg/L. The EC50 for yield inhibition (72 h EYC50) was 2.8 µg/L with a 95 % confidence interval ranging from 2.4 to 3.2 µg/L. The 72 h NOEC values for growth rate and yield inhibition were 1.6 and 0.47 µg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

3.3 µg/L

Additional information

The acute toxicity of the test material to algae 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).

Pseudokirchneriella subcapitata was exposed to the test material for 72 hours under static conditions. The test material was 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 exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to 0.010, 0.022, 0.046, 0.10, 0.22, 0.46 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.

Samples taken from all groups were analysed freshly. The actual concentrations were at the level of 66 to 98 % of nominal at the start of the test but decreased to 0.0049 to 1.4 % of initially measured at the end of the test. Based on these results, the time weighted average (TWA) concentrations were calculated to be 0.97, 0.22, 0.47, 1.6, 4.5, 13 and 76 µg/L at nominal concentrations of 0.010, 0.022, 0.046, 0.10, 0.22, 0.46 and 1.0 mg/L, respectively.

A very sharp increase of toxicity was observed at the TWA concentration of 4.5 µg/L for both growth rate and yield inhibition. Below this concentration the observed effects were low, whereas at 4.5 µg/L and above, nearly total inhibition was observed. Statistically significant inhibition of growth rate was found at test concentrations of 4.5 µg/L and higher, whereas for yield was statistically significantly inhibited at 1.6 µg/L and higher. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 4.5 µg/L when compared to the control. The study met the acceptability and was considered valid.

Under the conditions of this study, the test material inhibited growth rate significantly at a TWA concentration of 4.5 µg/L and higher.

The EC50 for growth rate inhibition (72 h ERC50) was 3.3 µg/L with a 95 % confidence interval ranging from 3.0 to 3.7 µg/L. The EC50 for yield inhibition (72 h EYC50) was 2.8 µg/L with a 95 % confidence interval ranging from 2.4 to 3.2 µg/L. The 72 h NOEC values for growth rate and yield inhibition were 1.6 and 0.47 µg/L, respectively.