1,3-di-o-tolylguanidine

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:

from may 2012 to august 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

DIORTHOTOLYL GUANIDINE was measured at all concentrations at the beginning and the end of the test (in non-inoculated test solutions (without algae) at the highest concentration tested).
Storage of samples: None
Samples treatment: Dilution 2 to 20 in acetonitrile.

Vehicle:

no

Details on test solutions:

For the range finding test and for the definitive test a stock parent solution at 100 mg/L of the test item was prepared the day when starting the test by mixing 100 mg of the test item DIORTHOTOLYL GUANIDINE in 1 liter of dilution water. This solution was stirred during 1.5 hour, then filtered through a cellulose ester Millipore filter 0.45µm (HAWP 04700) in order to obtain a clear solution. This solution was used to prepare the convenient range of nominal concentrations: 100, 50, 10, 5, 1, 0.5 and 0.1 mg/L.

For the definitive test a stock parent solution at 100 mg/L of the test item was prepared the day when starting the test by mixing 100 mg of the test item DIORTHOTOLYL GUANIDINE in 1 liter of dilution water. This solution was stirring during 1.5 hour and was filtered through a cellulose ester Millipore filter 0.45µm (HAWP 04700) in order to obtain a clear solution. This solution was used to prepare the convenient range of nominal concentrations: 50, 26, 13.6, 7, 3.7, 1.92 and 1 mg/L

Test organisms (species):

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

Details on test organisms:

Pseudokirchneriella subcapitata, CCAP 278/4 stock (previously named Raphidocelis subcapitata and Selenastrum capricornutum) are obtained from the Culture Centre of Algae and Protozoa (Ambleside, UK).
The conditions used for culturing algae are described in Annex 2 of the OECD 201 guideline. Two flasks, each containing approximately 100 mL of axenic stock culture of algae are incubated at 23 ± 1 °C under lighting (photoperiod: 16 hours of illumination, 8 hours of darkness), slowly continuously shaken. These stock cultures are renewed every week, using two new cultures.

The quality of the stock culture was verified for the absence of micro-organisms and deformed cells under microscopic observation before use. Three days before the beginning of the study two pre-cultures were prepared by inoculating each stock suspension of algae (5 mL) into sterile dilution water (500 mL). The pre-cultures were incubated under the same conditions as those used for the stock cultures. Only one of the two pre-cultures was used to inoculate the test flasks; the second one was to be used only if the first one was damaged.

At the beginning of the test, the cell concentration of the pre-culture was determined. The result was used to calculate the volume to be introduced into each test flask in order to get an initial cell concentration of 10^4 cells/mL. The cell density of the pre-culture was about 1.83 x 10^6 cells/mL for the preliminary test and about 1.48 x 10^6 cells/mL for the definitive test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No post-exposure observation period was done.

Test temperature:

23°C +/- 1°C

pH:

See "Any other information results incl. tables"

Dissolved oxygen:

See "Any other information results incl. tables"

Salinity:

Test was performed in freshwater.

Nominal and measured concentrations:

See "Any other information results incl. tables"

Details on test conditions:

The incubation was performed in a phytoculture cabinet that allows test flasks to be incubated under precise conditions: temperature was set to 23 ± 1°C ; flasks were continuously shaken with a rotation at 20 rpm and constantly illuminated by 8 fluorescent tubes between 6,000 and 10,000 lux (Mazdafluor, white industry 33).
The study was performed using 120 mL glass bottles stoppered with cellulose bungs. The filling volume was 50 mL.
Physico-chemical parameters were measured using a METTLER TOLEDO 345 pH meter for measurement of pH and with a WTW OXI 538 oxymeter for dissolved oxygen measurement. Determination of algae concentration was carried out using cytofluorimetry with a Cytofluor 2350 device in the range-finding test. In the definitive test, cells are numbered with a cell counter (Beckman Coulter Z2).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL (1.1 - 4.6)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL (3.3 - 9.5)

Details on results:

The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear, colourless over the period of the test. No precipitation was observed at the end of the test.
Microscopic observation confirmed that the algae appeared normal at the end of the test: the normal shape of P. subcapitata algae is a crescent shaped cell with an average length of 5-10 µm. Dissolved oxygen and pH were measured in the test solutions at the beginning and at the end of the test
and are presented in Table below. Measurements were carried out in blank non-inoculated solution at T0 and T72 and in control inoculated solution at T72.

Results with reference substance (positive control):

The sensitivity of the test system and the methodology are evaluated every two months by performing an algal growth inhibition test on potassium dichromate. The nearest values of ErC50 and EbC50 obtained on June 1 2012 were respectively 0.72 mg/L and 0.54 mg/L. For information, ISO 8692 reports the following results for an inter-laboratory exercise on potassium dichromate: ErC50: 0.60 to 1.03 mg/L EbC50: 0.20 to 0.75 mg/L.

Measured pH and O₂concentrations in the different test systems

nominal conc. mg/L	pH		Dissolved O2 (mg/L)
	T0	T72h	T0	T72h
0 (BI)	7.97	7.97	8.3	8.5
0 (T)	-	10.30	-	13.0
1.00 (g)	8.01	9.18	8.4	10.1
1.92 (f)	8.02	9.0	8.4	10.0
3.68 (e)	8.07	8.77	8.4	9.9
7.06 (d)	8.19	8.38	8.3	8.6
13.56 (c)	8.44	8.06	8.2	8.6
26.04 (b)	8.75	8.06	8.2	8.6
50.0 (a)	9.11	8.11	8.1	8.5

Average cell densities, biomass (A) and growth rates (m)

nominal conc. mg/L	Average cell density (cell/mL)				A	µ
	T0	T24h	T48h	T72h
0 (T)	1.00E+04	9.33E+04	2.36E+05	1.03E+06	103	1.546
1.00 (g)	1.00E+04	7.69E+04	2.42E+05	1.15E+06	115	1.580
1.92 (f)	1.00E+04	5.69E+04	2.35E+05	1.08E+06	108	1.561
3.68 (e)	1.00E+04	6.10E+04	2.20E+05	8.41E+05	84	1.477
7.06 (d)	1.00E+04	4.65E+04	1.58E+05	3.92E+05	39	1.223
13.56 (c)	1.00E+04	3.35E+03	6.68E+03	5.65E+03	1	-0.190
26.04 (b)	1.00E+04	4.15E+03	4.55E+03	4.12E+03	0	-0.296
50.0 (a)	1.00E+04	4.42E+03	5.25E+03	3.42E+03	0	-0.358

 

Definitive test – Average percentage inhibition of cell growth (IAi) and growth rate (Iµi)

nominal conc. mg/L	IAi	Iµi
0 (T)	0.00	0.00
1.00 (g)	-5.61	-2.23
1.92 (f)	1.62	-0.99
3.68 (e)	17.57	4.43
7.06 (d)	54.30	20.89
13.56 (c)	100.00	112.31
26.04 (b)	100.00	119.14
50.0 (a)	100.00	123.16

Concentration of test item

nominal conc. (mg/L)	Measured in non inoculated solutions (mg/L)
	Initial	Final**	Final/Initial
0 (Bl)	< LD	< LD	-
1.00 (g)	< LQ	0.93 (0.87)	103.3
1.92 (f)	1.71	1.76 (1.66)	102.9
3.68 (e)	3.25	3.31 (3.18)	101.8
7.06 (d)	6.16	6.13 (6.03)	99.5
13.56 (c)	11.8	11.7 (11.6)	99.2
26.04 (b)	22.5	21.9	97.3
50.0 (a)	42.8	44.1	103.0
100 (stock solution)	84.8	-	-

< DL : concentration lower than the Detection Limit of the analytical method (0.047 mg/L).

< QL : concentration lower than the Quantification Limit of the analytical method (0.16 mg/L).

** Within parenthesis: concentrations of measured in inoculated series (with algae).

Validity criteria fulfilled:

yes

Remarks:

See "Overall remarks"

Conclusions:

The determination of the growth inhibition of the freshwater algae Pseudokirchneriella subcapitata (previously known as Raphidocelis subcapitata and Selenastrum capricornutum) exposed to the test item for a duration of 72 hours was assessed according to the OECD Guideline 201 under GLP.

Executive summary:

The determination of the growth inhibition of the freshwater algaePseudokirchneriella subcapitata (previously known asRaphidocelis subcapitata and Selenastrum capricornutum) exposed to the test item DIORTHOTOLYL GUANIDINE for a duration of 72 hours was assessed according to the OECD Guideline 201.

Algae were exposed to 50 to 1 mg/L of DIORTHOTOLYL GUANIDINE dissolved in dilution water. The toxic effect measured during the assay was the inhibition of cellular multiplication over a time period of 72 hours. The concentrations of test item causing a 50% reduction in biomass (EbC50) and in growth rate (ErC50) were estimated. EC10were also estimated. The results were as follows (values expressed in nominal concentrations):

 

 

End point	EC50-72h (mg/L) (95% CI)	EC10-72h (mg/L) (95% CL)
Growth rate – Cell multiplication (r)	7.2 (ND)	4.8 (ND)

CI: confidence interval ; ND: not determined

Description of key information

Two guideline studies available with results in the same range.
Key results used for the CSA (Gancet et al., 2012):
72h-Erc50DOTG = 7.2 mg/L.
72h-Erc10DOTG = 4.8 mg/L.
Study performed in accordance with:
- OECD testing guideline 201 and 
- GLP requirements. 

Key value for chemical safety assessment

EC50 for freshwater algae:

7.2 mg/L

EC10 or NOEC for freshwater algae:

4.8 mg/L

Additional information

The determination of the growth inhibition of the freshwater algaePseudokirchneriella subcapitata (previously known as Raphidocelis subcapitata and Selenastrum capricornutum) exposed to the test item DIORTHOTOLYL GUANIDINE for a duration of 72 hours was assessed according to the OECD Guideline 201.

Algae were exposed to 50 to 1 mg/L of DIORTHOTOLYL GUANIDINE dissolved in dilution water. The toxic effect measured during the assay was the inhibition of cellular multiplication over a time period of 72 hours. The concentrations of test item causing a 50% reduction in biomass (EbC50) and in growth rate (ErC50) were estimated. EC10were also estimated. The results were as follows (values expressed in nominal concentrations):

 

 

End point	EC50-72h (mg/L) (95% CI)	EC10-72h (mg/L) (95% CL)
Cell growth – Biomass (b)	5.5 (3.3-9.5)	3 (1.1-4.6)
Growth rate – Cell multiplication (r)	7.2 (ND)	4.8 (ND)

CI: confidence interval ; ND: not determined

 

The toxicity of DOTG to the inhibition of Selenastrum Capricornutum was also assessed according to the OECD 201 guideline, in a GLP study performed by the Japanese Minstry of Environment.

Water quality parameters of temperature, dissolved oxygen and pH were within acceptable limits.

The results of the 72 hours semi-static inhibition toxicity study are summarized below:

DOTG 72h-ECb50 = 5.56 mg/L (measured value).

DOTG 72h-ECb10 = 2.27 mg/L (measured value).

Another Two guideline studies available with results in the same range.
Key results used for the CSA (Gancet et al., 2012):