Trisodium trihydrogen 5,5',5''-(1,3,5-triazine-2,4,6-triyltriimino)tris[5-sulphonatosalicylate]

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017 01 31 to 2017-02-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD and GLP guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium trihydrogen 5,5',5''-(1,3,5-triazine-2,4,6-triyltriimino)tris[5-sulphonatosalicylate]

EC Number:

279-091-6

EC Name:

Trisodium trihydrogen 5,5',5''-(1,3,5-triazine-2,4,6-triyltriimino)tris[5-sulphonatosalicylate]

Cas Number:

79135-90-3

Molecular formula:

C24H18N6O18S3.3Na

IUPAC Name:

trisodium 3-({4,6-bis[(3-carboxy-2-hydroxy-5-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)-5-carboxy-4-hydroxybenzene-1-sulfonate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
The limit concentration and the control were analytically verified via UPLC-DAD at the start of the exposure (0 hours) and at the end of the exposure (48 hours). The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).

Sampling for the analytical monitoring
At the start of the exposure (0 hours), samples of the fresh media were taken after preparation of the limit concentration and analyzed.
At the end of the exposure (48 hours), samples of the old media were taken directly from the test vessels and analyzed.

Range (target)
Recoveries of the test item should be within ± 20% of the nominal or initially measured concentration.

Test solutions

Vehicle:

no

Details on test solutions:

Limit concentration
A limit concentration of 126 mg/L of the test item, which corresponds to a nominal concentration of 100 mg/L of the main component, was tested.
The limit concentration was selected based on the results of a non-GLP preliminary range finding test under static conditions.

Preparation of the limit concentration
The limit concentration (126 mg/L of the test item were weighed out) was freshly prepared with dilution water before the start of the exposure (0 hours). The limit concentration was mixed thoroughly by manual agitation until the test item was completely dissolved.

Control
Dilution water without test item incubated under the same conditions as the test groups.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system
Daphnia magna STRAUS (Clone 5)

Reason for the selectionof the test system
Daphnia magna is the preferred species in accordance with the
test guideline and is bred at the test facility.

Origin
Institut für Wasser-, Boden- und Lufthygiene (WaBoLu),
14195 Berlin, Germany

Breeder
Noack Laboratorien GmbH,
Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture
In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2 °C, in an incubator, 16 hours illumination; light intensity of max. 1500 lx

Culture medium
Elendt M4, according to OECD 202, Annex 3 (2004), modified to a total hardness of 160 to 180 mg CaCO3/L, is used.

Culture feeding
The culture daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL. The algae were cultured at the test facility.

Origin of the food algae
Sammlung von Algenkulturen (SAG),
Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

Total hardness [mg CaCO3/L] : 162

Test temperature:

20.6 °C

pH:

Water Quality Parameters at the Start of the Exposure (0 hours)
(measured in one additional replicate (without daphnids) of the limit concentration and the control)
Nominal
test item concentration
[mg/L] main component
[mg/L] pH-value Dissolved
O2 concentration [mg/L]
126 100 7.61 8.69
Control Control 7.72 9.32

Water Quality Parameters at the End of the Exposure (48 hours)
(measured in all replicates (containing daphnids) of the limit concentration and the control)
Nominal
test item concentration
[mg/L] main component
[mg/L] pH-values Dissolved O2 concentration [mg/L]
Replicates Replicates
1 2 3 4 1 2 3 4
126 100 7.48 7.47 7.51 7.49 8.04 8.14 7.99 7.80
Control Control 7.61 7.62 7.22 7.46 7.41 7.22 7.49 7.64

Dissolved oxygen:

see above

Conductivity:

2017-01-31 [µS/cm] : 420

Nominal and measured concentrations:

Nominal: 1.00 mg/L
Initial measured 0.407 mg/L

Details on test conditions:

Test method
The study was performed with a static test design.

Test duration
48 hours

Test vessels
Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses

Test volume
20 mL in each test replicate (see ‘Application’)

Dilution water
Same as culture medium

Number of daphnids and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids, were used for the saturated solution and the control.

Age of the daphnids
Less than 24 hours old daphnids from a healthy stock were used for at the start of the exposure the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Acclimatization
Acclimatization of the daphnids was not necessary, because the dilution water was equivalent to the culture medium.
 
Application
20 g test solution per replicate was weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water by a pipette.

Test temperature (target)
18 - 22 °C, constant within ± 1 °C

Illumination (target)
Diffuse light, light intensity of max. 1500 lx

Photoperiod (target)
16/8 hours light/dark cycle

Feeding
The daphnids were not fed during the study.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

other: EC 10/50/100

Effect conc.:

> 126 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

other: EC 10/50/100

Effect conc.:

> 126 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2017 01-12 to 2017-01-13.

EC50-Value (with 95% confidents limits) of the Reference Item Potassium dichromate
based on nominal concentrations mg/L, (0 - 24 hours)
Current Study Valid Range
EC50 1.92 mg/L
0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5
0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A
95% confidence limits 1.09 - 3.32 mg/L

Reported statistics and error estimates:

Methods of evaluation
The effects of the limit concentration were empirically derived from the observed immobilization rates.
Since the measured concentrations of the test item were within ± 20% of the nominal concentration, the effect concentrations (EC10 / 50 / 100) were based on the nominal concentration of the test item.

EC-values and statistical analyses for reference item
An EC50-value was calculated for the reference item by sigmoidal dose-response regression. The respective 95% confidence limits were calculated from the standard error and the t distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism5.

Software
All data were computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
- GraphPad Prism5, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

Biological Data

 

The percentage of immobility, determined in the limit concentration and in the control after 24 and 48 hours of exposure under static conditions, is given in the Table. The absolute numbers of immobile daphnids are presented in Table below.

 

Immobilization Rates after 24 and 48 hours of Exposure in the Definitive Test

(n = 20, divided into 4 replicates with 5 daphnids each)

Nominal concentration of the		IMMOBILIZATION [%]
		24 hours					48 hours
test item   [mg/L]	main component [mg/L]	Replicates					Replicates
		1	2	3	4	MV	1	2	3	4	MV
126	100	0	0	0	0	0	0	0	0	0	0
Control		0	0	0	0	0	0	0	0	0	0

Absolute Numbers of immobileDaphnids after 24 and 48 hours ofExposure in the Definitive Test

(n = 20, divided into 4 replicates with 5 daphnids each)

Nominal concentration of the		Number of immobile Daphnids / Total number of Daphnids
		24 hours					48 hours
test item   [mg/L]	main component [mg/L]	Replicates					Replicates
		1	2	3	4	MV	1	2	3	4	MV
126	100	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
Control		0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20

 

The EC_{10 / 50 / 100}-values were based on the nominal concentration of the test item. For results, see Table1.

Additional Observations during the Definitive Test

 

Thelimit concentrationwas slightly brownish and visually clear during the exposure period.

 

 

Measured Exposure Concentrations during the Definitive Test

 

The concentration of the test item was analytically verified by UPLC-DAD at the start (0 hours) and at the end of exposure (48 hours) at the limit concentration and the control.

The measured concentration of the test item at the start of the exposure (0 hours) was 101% of the nominal value at the limit concentration level. The measured concentrations at the end of the exposure (48 hours) was also 101% of the nominal value.

The measured concentrations of the test item were within ± 20% of the nominal concentration. This indicates that the test item concentration was successfully maintained for the duration of the test.

 Measured Concentrations of the Test Item during the Definitive Test

Sampling date:		2017-01-31 Start of the exposure, 0 hours			2017-02-02 End of the exposure, 48 hours
Start of analysis:		2017-01-31			2017-02-02
Nominal concentration of the		test item
test item   [mg/L]	main component [mg/L]	Meas. conc. of the test item [mg/L]	Meas. conc. of the main component [mg/L]	%	Meas. conc. of the test item [mg/L]	Meas. conc. of the main component [mg/L]	%
126	100	127	101	101	128	101	101
Control		< LOQ			< LOQ

Meas. conc.    =measured concentration of the test item / main component of the test item, 1 injection,
  dilution factor taken into account

%                     =percent of the nominal concentrationof the test item / main component of the test item

LOQ                =limit of quantification of the analytical method (20.0 mg/L of the test item)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

At the limit concentration of 126 mg/L of the test item, which corresponds to a nominal concentration of 100 mg/L of the main component, no effects on Daphnia magna were observed.

Executive summary:

In the acute immobilization test to Daphnia magna (STRAUS), the effects of the limit concentration of 126 mg/L of the test item (batch number:CHA0 127492), which corresponds to 100 mg/L of the main component, were determined at the test facility from 2017‑01‑31 to 2017-02-02 according to OECD 202 (2004).

The study was conducted under static conditions over a period of 48 hours. The limit concentration was slightly brownish and visually clear during the exposure period.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to the limit concentration and the control.

The concentration of the test item was analytically verified by UPLC-DAD at the start (0 hours) and at the end of exposure (48 hours) at the limit concentration and the control. Details of the analytical method are presented in section12.

The measured concentration of the test item at the start of the exposure (0 hours) was 101% of the nominal value at the limit concentration level. The measured concentrations at the end of the exposure (48 hours) was also 101% of the nominal value.

The measured concentrations of the test item were within ± 20% of the nominal concentration. This indicates that the test item concentration was successfully maintained for the duration of the test. Therefore, all effect concentrations given are based on the nominal concentration of the test item Anodal SH-1 hc dried.

The validity criteria of the test guideline were fulfilled.

 

At the limit concentration of 126 mg/Lof the test item, which corresponds to a nominal concentration of 100 mg/L of the main component, no effects on Daphnia magna were observed.

 

 

 

EC₁₀-, EC₅₀-and EC₁₀₀-Values

(based on the nominal concentration of the test item)

Effect concentrations	Test duration [hours]	Toxicity endpoint based on the nominal concentration of the test item [mg/L]
EC10 / 50 / 100	24	> 126
	48	> 126