[1,2,4]Triazolo[1,5-c]pyrimidine, 2,2'-dithiobis[5-ethoxy-7-fluoro-

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

Study period:

8 November 1993 to 13 November 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. No analytical determination of test substance concentration.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: 40 CFR Part 797 Subpart B

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

5-ethoxy-2-({5-ethoxy-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl}disulfanyl)-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidine

EC Number:

695-187-4

Cas Number:

166524-75-0

Molecular formula:

C14H12F2N8O2S2

IUPAC Name:

5-ethoxy-2-({5-ethoxy-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl}disulfanyl)-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidine

Test material form:

other: solid (unspecified)

Details on test material:

- Appearance: light beige solid

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

The test material was used as received; it was added directly to the test medium.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 1648
Stock cultures of this organism were maintained axenically by weekly transfer into sterile algal assay medium (AAM).
- Culturing media and conditions: same as test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

5 d

Test conditions

Test temperature:

24.1 - 24.7 °C

pH:

7.2 - 7.5 (without algae); 7.6 - 8.4 (with algae)

Nominal and measured concentrations:

0, 15.75, 31.5, 63, 126, 252 and 504 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: sterilised 250 mL borosilicate Erlenmeyer flasks
- Type: closed with Shimadzu closures
- Fill volume: 100 mL (algal test medium)
- Aeration: flasks were continuously shaken at 100 rpm
- Initial cell density: approximately 10 000 cells/mL. Each test flask was dosed with 1 mL algal inoculum.
- No. of vessels per concentration (replicates): three. A counting blank was included with each test concentration (contained growth medium and the appropriate amount of test material but no algae)
- No. of vessels per control (replicates): six

GROWTH MEDIUM
- Standard medium used: yes (Algal Assay Medium) The culture medium used for the definitive test was the same as that used to maintain the stock culture except that the medium used in the toxicity tests did not contain the chelant, Na2EDTA.2H2O
- Preparation of the Algal Assay Medium:
Each stock solution was filtered through a 0.45 µm membrane filter, then stored at 4 °C; the solutions were brought up to room temperature before using. 1 mL of each stock solution A, B1, B2, B3 and D, plus 10 mL of C were added to 900 mL of dilution water. The mixture was then made up to 1 L with additional water.
A: 12.75 g NaNO3, 6.08 g MgCl2.6H2O and 2.20 g CaCl2.2H2O was added to 500 mL dilution water
B1: 7.35 g MgSO4.7H2O dissolved in 500 mL dilution water
B2: 7.5 g NaHCO3 dissolved in 500 mL dilution water
B3: 0.522 g K2PO4 dissolved in 500 mL dilution water
C: 5 mL of number 1 (below) and 5 mL of number 3 (below) diluted to 500 mL with sterile dilution water.
1: 1.86 g H3BO3, 4.17 g MnCl2.4H2O, 0.0327 g ZnCl2 and 0.0726 g NaMoO4.2H2O dissolved in 1000 mL dilution water
2: 2.86 g CoCl2.6H2O and 0.22 g CuCl2.2H2O dissolved in 1000 mL dilution water
3: 2.5 mL of number 2 was made up to 500 mL
D: 0.30 g Na2EDTA.2H2O and 0.16 g FeCl3.6H2O dissolved in 1000 mL dilution water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous
- Light intensity and quality: 3800 - 5050 lux

EFFECT PARAMETERS MEASURED
The algal densities of the inoculum and test cultures were determined by electron particle counting using a Coulter Multisizer. Total cell counts were determined at approximately 24, 48, 72, 96 and 120 hours. Cells were cumulatively counted from a lower threshold equivalent spherical diameter of approximately 2.7 µm to a higher threshold equivalent spherical diameter of approximately 8.6 µm. The background particle count (growth medium and test material) was concurrently subtracted from that of the sample dilution (growth medium, test material and algae) to obtain cell density. A blank correction for the isotonic saline solution used in preparing the dilutions was also compensated for in the calculations.

PHYSICAL ANALYSIS
Extra flasks without algae were added to the test for each nominal concentration to monitor pH at the initiation of the test and every day thereafter. At the end of the test a final pH was taken in a replicate test flask at each test concentration with algae. The temperature of a representative test solution was continuously monitored during the test. The light intensity was checked on a daily basis with readings taken corresponding to the positions of the test flasks in the incubator.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: test concentrations were set in a geometric series with a progression factor of 2.0
- Range finding study: yes
- Test concentrations: 0.1, 1.0, 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: yes; the results indicated essentially no effect at any concentration in comparison to the controls.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

259 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.0 - 534.8 mg/L (95 % CL)

Effect concentrations 2

Duration:

120 h

Dose descriptor:

EC50

Effect conc.:

364 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 175.4 - 552.7 mg/L (95 % CL)

Effect concentrations 3

Duration:

120 h

Dose descriptor:

NOEC

Effect conc.:

126 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

During the first three days of the study, the data for the 252 mg/L concentration showed no algal growth. This was probably due to the fact that the test material was insoluble at concentrations above 126 mg/L. At the 252 mg/L concentration, the precipitate particle size appeared to be in the same range as the algal cell size and therefore produced a large background count. As the algal growth exceeded the background count, the effects of the test material particle size was reversed and a positive algal growth rate was observed.

The 72 and 120 hour EC50 values (and their 95 % confidence intervals) for algal growth were 259 (0.0 - 534.8) and 364 (175.4 - 552.7) mg/L, respectively, and for percent inhibition were 310 (5.1 - ∞) and 140 (18.1 - 1080.3) mg/L, respectively. The 120 hour NOEC for algal growth was 126 mg/L.

Reported statistics and error estimates:

The EC10, EC50 and EC90 values for algal growth were numerically derived based on calculated regression equations for each 24 hour period. The no observed effect concentrations (NOECs) for algal growth were calculated using analysis of variance and the Dunnett's test. The percent inhibition values were calculated by regression based on the comparison of the areas under the growth curves between the control group and each exposure concentration.

Any other information on results incl. tables

Table 1: Mean Values for Growth for Each Replicate Flask

Nominal Conc. (mg/L)	Day 1	Day 2	Day 3	Day 4	Day 5
0 (control)	21840	67615	371400	918800	2009333
	20007	72099	298333	895733	2274933
	25927	72099	285867	946133	2540933
	23733	67691	285400	822200	2178533
	24933	72960	318267	894800	2236533
	21647	80231	357933	779467	2109200
15.8	40787	89072	306067	826400	2254533
	37320	88971	284000	855200	2099467
	29927	96596	285800	910933	2052667
31.5	26627	79065	274067	839733	2154933
	25007	80180	247000	855600	1870267
	29953	144957	327400	1161200	2693467
63	39100	60749	433000	1076533	3102400
	30133	75721	308800	749733	2152933
	32993	89908	293000	809333	2113333
126	53640	50971	336333	784400	1899600
	45373	50236	283067	720400	1955200
	40393	75164	330267	789200	2306667
252	0	0	43067	604000	1543333
	0	0	5067	669467	1546400
	0	0	0	788133	1701200
504	61613	95481	24867	120933	583467
	48553	0*	0	94133	457867
	73807	60065	146467	216667	511600

* All negative numbers in the data were reset to zero for statistical purposes

Table 2: Results Based on Growth Rate

Time point	Results	EC10 (mg/L)	EC50 (mg/L)	EC90 (mg/L)	NOEC (mg/L)
24 hours	Total cell count (cells/mL)	0	0	0	126
	95% CL	(0.0 - 745.1)	(0.0 - 616.4)	(0.0 - 529.1)
48 hours	Total cell count (cells/mL)	82	316	1089	126
	95% CL	(3.2 - 2085.7)	(11.3 - 8820.3)	(29.9 - ∞)
72 hours	Total cell count (cells/mL)	53	259	458	126
	95% CL	(0.0 - 326.1)	(0.0 - 534.8)	(163.9 - 751.1)
96 hours	Total cell count (cells/mL)	101	346	573	126
	95% CL	(0.0 - 265.2)	(175.6 - 515.8)	(385.8 - 760.3)
120 hours	Total cell count (cells/mL)	103	364	606	126
	95% CL	(0.0 - 284.3)	(175.4 - 552.7)	(395.8 - 815.8)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 72 and 120 hour EC50 values (and their 95 % confidence intervals) for algal growth were 259 (0.0 - 534.8) and 364 (175.4 - 552.7) mg/L, respectively, and for percent inhibition were 310 (5.1 - ∞) and 140 (18.1 - 1080.3) mg/L, respectively. The 120 hour NOEC for algal growth was 126 mg/L.

Executive summary:

The toxicity of the test material to aquatic algae was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 201 and 40 CFR Part 797 Subpart B.

During the study, the freshwater green alga Selenastrum capricornutum Printz was exposed to the test material, at nominal concentrations of 0 (control), 15.75, 31.5, 63, 126, 252 and 504 mg/L, for 5 days. The EC50 values, together with the NOEC, were based on nominal concentrations.

Under the conditions of this study, the 72 and 120 hour EC50 values (and their 95 % confidence intervals) for algal growth were 259 (0.0 - 534.8) and 364 (175.4 - 552.7) mg/L, respectively, and for percent inhibition were 310 (5.1 - ∞) and 140 (18.1 - 1080.3) mg/L, respectively. The 120 hour NOEC for algal growth was 126 mg/L. Results are based on nominal test concentrations.