6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The concentration of NaHCO3 was increased to be twice of that indicated in the Guideline 201. This concentration has been found to be optimal

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione

EC Number:

249-682-3

EC Name:

6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione

Cas Number:

29529-99-5

Molecular formula:

C11H20N4S2

IUPAC Name:

6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Specimens were drawn from freshly prepared treatments and those incubated for 72 hr. The supporting analysis of the specimens could not be performed on the same day of collection of the specimens. Therefore, all specimens from freshly prepared test solutions and those incubated for 72hrs were stored deep-frozen at ≤ -18°C in glass bottles and analysed after thawing. Before analysis the treatments were diluted as follows: 1 mg/L specimens without dilution, 3.2 mg/L specimens werde diluted 1:2, 10.2 mg/L specimens were diluted 1:10, 32.8 mg/L specimens were diluted 1:20, and 104.9 mg/L specimens were diluted 1:100 using a mixture of 900 mL 0.2 mM ammoniumacetate + 100 mL acetonitrile being spiked 0.1% with formic acid.

Test solutions

Vehicle:

not specified

Details on test solutions:

For the main test, the following amounts of suspensions with the test item were prepared as described above:
[1] 1.25 mg / L ultrapure water (resulting 1.0 mg/L final nominal conc. [“WAF”])
[2] 4.00 mg / L ultrapure water (resulting 3.2 mg/L final nominal conc. [“WAF”])
[3] 12.80 mg / L ultrapure water (resulting 10.2 mg/L final nominal conc. [“WAF”])
[4] 40.96 mg / L ultrapure water (resulting 32.8 mg/L final nominal conc. [“WAF”])
[5] 131.07 mg / L ultrapure water (resulting 104.9 mg/L final nominal conc. [“WAF”])

Solutions a), c), e), g), i), k) were prepared in 7 replicates, and solutions d), f), h), j) and l) were prepared in triplicate; solution b) was prepared as a single solution.
The incubation of the main culture took place under the same conditions as the pre-culture. The algae were held in suspension by stirring for 15 min per hour on a magnetic stirrer.
By addition of pre-culture medium, the absorbance E578 nm of the grown pre-culture was diluted, so that the initial algal cell concentration at t0 in the test solutions (control) was 3*103 cells/mL.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

As a functional control, the biomass production of the algae in the control solution was used. Within 72 hours the cell concentration (biomass) in the control cultures (tested in 7 replicate control solutions) should have increased by a factor of at least 16 (growth rate of 0.92/day) – a value 123.2 with a growth rate 1.601/d was met.

Further critera of validity are:
• The mean coefficient of variation for section-by-section specific growth rates at the different times of measurement in the control cultures must not exceed 35% - a value of 33.0 was met. This criterion applies to the mean value of coefficients of variation calculated for replicate control cultures.
• The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% (for Desmodesmus subspicatus) – a value 0.6 % was met.
• Increase in pH in the controls without any test item should have been ≤ 1.5 within 72 hr – the increase measured was 1.43 units. 1.43 units are in the requested range of the Guideline OECD 201.

Study design

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

The unicellular algae Desmodesmus subspicatus were exposed to different concentrations of the test item for 72 hours at 23.3 to 23.9°C at constant illumination.

Post exposure observation period:

not specified

Test conditions

Hardness:

not specified

Test temperature:

of 23.2 – 23.9 °C

pH:

7.18- 8.12

Dissolved oxygen:

not specified

Salinity:

not specified

Conductivity:

not specified

Nominal and measured concentrations:

[1] 1.25 mg / L ultrapure water (resulting 1.0 mg/L final nominal conc. [“WAF”])
[2] 4.00 mg / L ultrapure water (resulting 3.2 mg/L final nominal conc. [“WAF”])
[3] 12.80 mg / L ultrapure water (resulting 10.2 mg/L final nominal conc. [“WAF”])
[4] 40.96 mg / L ultrapure water (resulting 32.8 mg/L final nominal conc. [“WAF”])
[5] 131.07 mg / L ultrapure water (resulting 104.9 mg/L final nominal conc. [“WAF”])

Reference substance (positive control):

not specified

Results and discussion

Effect concentrations

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 104.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

On Basis of the nominal Concentrations (“WAF”)
[mg 6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione / L]
Yield (0 - 72hr)
EL10 0.22
95%-CL lower 0.09
upper 0.23
EL20 0.75
95%-CL lower 0.32
upper 1.81
EL50 8.11
95%-CL lower 2.85
upper 23.59
Yield LOEL ≤ 1.00
NOEL < 1.00
Section-by-section growth rate (0-72hr)
EL10 0.62
95%-CL lower 0.23
upper 1.64
EL20 2.20
95%-CL lower 0.85
upper 5.79
EL50 25.07
95%-CL lower 7.56
upper 80.45
LOEL ≤ 1.00
NOEL < 1.00
Growth rate (0 - 72 hr)
EL10 2.23
95%-CL lower 0.83
upper 6.03
EL20 27.57
95%-CL lower 9.32
upper 85.31
EL50 n.d. (>104.9)
95%-CL lower n.d. (>104.9)
upper n.d. (>104.9)
Growth rate LOEL ≤ 1.00
NOEL < 1.00
n.d.: not determined due to mathematical reasons or inappropriate data

Any other information on results incl. tables

Inhibition Values

NominalConcen­tra­tion(“WAF”)of the Test Item [mg/L]*	Growth Rates (day^-1)	Inhibition of the Growth Rate after 72hr [%]	Yield after 72hr^1)	Inhibition of the Yield after 72hr [%]
Control (0)	1.792	-	5.7*10^5	-
1.0	1.740	2.9	4.9*10^5	14.4
3.2	1.646	8.2	3.7*10^5	34.6
10.2	1.456	18.8	2.1*10^5	63.8
32.8	1.395	22.2	1.7*10^5	69.9
104.9	1.377	23.3	1.6*10^5	71.6

 

¹⁾Yield = Cell number at time t_72hr– cell number at time t₀;²⁾negative value indicates an activation

* Concentrations as active substance

The complete statistical evaluation of all test results obtained within the definitive main test is given in the statistical appendix of this report.

Individual cell numbers of all tests are presented in chapter 15.4 (appendix) of this report.

 

 

 

 

Results of the Analytical Phase

The results of the supporting analyses can be summarised as follows:

Nominal Concentration of Test Item  6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione (“WAF”) [mg/L]	mg/L Test Item   (“WAF”) t0	mg/L Test Item   (“WAF”) t72hr	% of t0	Mean*^)measured Concentration of Test Item [mg/L]
0	0	0	-	0
1.0	1.098	1.114	101.4	1.105
3.2	3.348	3.337	99.7	3.342
10.2	10.238	10.348	101.1	10.293
32.8	34.172	33.705	98.6	33.938
104.9	59.884	60.021	100.2	59.952

 

*⁾geometric means

As a result of the supporting analyse it can be stated that minor concentrations of the test item measured at the highest concentration may be due to “WAF”-preparation effects (adsorption to filters etc). Mean measured concentrations at 1.0 to 32.8 mg/l were all at 100.9 to 110.5 of the nominal concentrations. The mean measured concentration at 104.9 mg/L was at 57.2%of the nominal concentration. Furthermore,all measured concentrations remained stable >98% during incubation and thus, the results of the biological part should be based on the nominal concentrations applied because the necessary stability of ≥ 80% could be shown.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

On Basis of the nominal Concentrations(“WAF”)
[mg6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione/ L]

Yield (0 - 72hr)                                
                            EL10            0.22
         95%-CL    lower            0.09
                           upper            0.23 
                            EL20            0.75
         95%-CL    lower            0.32
                           upper            1.81 
                            EL50            8.11
         95%-CL    lower            2.85
                           upper          23.59 
               Yield   LOEL        ≤ 1.00
                           NOEL        < 1.00
Section-by-section growth rate (0-72hr)
                            EL10            0.62
         95%-CL    lower            0.23
                           upper            1.64
                            EL20            2.20
         95%-CL    lower            0.85
                           upper            5.79
Section-by-section growth rate (0-72hr)
                      EL50          25.07
   95%-CL    lower             7.56
                     upper           80.45
                          LOEL        ≤ 1.00
                           NOEL        < 1.00
Growth rate (0 - 72 hr)               
                     EL10            2.23
   95%-CL    lower            0.83
                     upper            6.03
                      EL20          27.57
   95%-CL    lower            9.32
                     upper         85.31
                      EL50n.d. (>104.9)
   95%-CL    lower n.d. (>104.9)
                     upper n.d. (>104.9)
    Growth rate   LOEL        ≤ 1.00
                           NOEL        < 1.00
n.d.: not determined due to mathematical reasons or inappropriate data

Executive summary:

The toxicity of 6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithionetowards algae was testedaccording to OECD-Guideline No. 201, in the Version dated 23-March-2006. The toxic effect was in­vestigated by determination of the inhibition of the growth rate of the algae and the yield during the exposure period of 72 hours. The test was performed using five concentrations of “WAFs” (Water accommodated fractions) in the nominal range of 1.0 to 104.9 mg/L of 6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione.The following EL-values (“Effective Loading”) were obtained:

 

On Basis of thenominal Concentrations(“WAF”) [mg6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione/ L]

Yield (0 - 72hr)                                                             EL10            0.22          95%-CL    lower            0.09                            upper            0.23                              EL20            0.75          95%-CL    lower            0.32                            upper            1.81                              EL50            8.11          95%-CL    lower            2.85                            upper          23.59                 Yield   LOEL        ≤ 1.00                            NOEL        < 1.00 Section-by-section growth rate[JM2] (0-72hr)                             EL10            0.62          95%-CL    lower            0.23                            upper            1.64                             EL20            2.20          95%-CL    lower            0.85                            upper            5.79

Section-by-section growth rate (0-72hr)                       EL50          25.07    95%-CL    lower             7.56                      upper           80.45                           LOEL        ≤ 1.00                            NOEL        < 1.00 Growth rate (0 - 72 hr)                                     EL10            2.23    95%-CL    lower            0.83                      upper            6.03                       EL20          27.57    95%-CL    lower            9.32                      upper         85.31                       EL50n.d. (>104.9)    95%-CL    lower n.d. (>104.9)                      upper n.d. (>104.9)     Growth rate   LOEL        ≤ 1.00                            NOEL        < 1.00 n.d.: not determined due to mathematical reasons or inappropriate data

 

As a result of the supporting analyse it can be stated that minor concentrations of the test item measured at the highest concentration are due to “WAF”-preparation. Nevertheless all measured concentrations remained stable during incubation and thus, the results of the biological part should be based on the nominal concentrations applied because the necessary stability of ≥ 80% could be shown.

The validity criteria for the study as given by the OECD Guideline 201 were all met and the study is therefore considered to bevalid.