6,6',6''-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoic acid, compound with 2,2',2''-nitrilotriethanol (1:3)

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study, acceptable for assessment

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Results of the harmful effects of water pollutants to Daphnia magna in the 21 day reproduction test.

Author:

Kuehn R, Pattard M, Pernak KD and Winter A

Year:

1989

Bibliographic source:

Water Research 23(4): 501-510.

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Provisional proposal by German Federal Environment Agency: Extended toxicity test using Daphnia magna (determination of NOEC for reproduction rate, mortality and the time of appearance of first offspring; 01 Jan 1984)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

other: This internal test method of the German Federal Environment Agency is a precursor of the OECD guidelines for the Daphnia reproduction test (initial version: OECD 202, part II; recent version: OECD 211)

Principles of method if other than guideline:

German title: Umweltbundesamt: Vorläufiger Testverfahrensvorschlag: Verlängerter Toxizitätstest bei Daphnia magna (Bestimmung der NOEC für Reproduktionsrate, Mortalität und den Zeitpunkt des ersten Auftretens von Nachkommen; 21 d). Stand: 01.01.1984", gegeben als "Empfehlung des Umweltbundesamtes zur Durchführung der Prüfung nach § 5 Abs. 1 Nr. 3 der Verordnung über Anmeldeunterlagen und Prüfnachweise nach dem ChemG.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2',2''-nitrilotriethanol

EC Number:

203-049-8

EC Name:

2,2',2''-nitrilotriethanol

Cas Number:

102-71-6

Molecular formula:

C6H15NO3

IUPAC Name:

2,2',2''-nitrilotriethanol

Details on test material:

- Name of test material (as cited in study report): Triethanolamine
- Analytical purity: no data

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling occasions: first sampling: on day of water exchange before 7th day (no offspring); second sampling: on day of water exchange between 16th and 21st day
- Concentrations: stock solution and selected dilution levels (in control and test concentrations without animals and food: fresh and aged after 48/72 h)
- Sampling method: not specified
- Sample storage conditions before analysis: not specified

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: stock solution (2000 mg/L), stirred for 24 h, dilution water heated to 30 °C to facilitate the solution process
- Controls: negative control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: waterfleas
- Strain/clone: IRCHA
- Justification for species other than prescribed by test guideline: not applicable
- Source: The stock culture was kept since 1982 at the testing laboratory under the conditions described below.
- culture: 
- 20-30 animals in 40 2-L beakers filled with >= 1.6 L tap water (hardness: 285.76 mg/L as CaCO3; pH 7.6-7.7; oxygen-saturated; water exchange: two times per week)
- beakers covered with watch glasses
- temperature: 20 °C      
- fluorescent light (9 h light : 15 h dark)      
- offspring removed daily from cultures      
- feeding: dried algae (Scenedesmus sp.; suspension: 9 g dried algae/L, 2 mL/beaker)
- Age of parental stock (mean and range, SD): no data
- Age of test animals: <= 24 h
- Feeding during test
- Food type: fish feed (Tetramin Hauptfutter; suspension of 200 g/L; day 1 to 7: 1 mL; day 8 to 21: 1.5 mL) and activated sludge from municpal sewage treatment plant (0.1 mL per day = COD 10 mg/L ± 15%)
- Amount: 15-20 mg COD/L per day
- Frequency: 3 times per week

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test conditions

Test temperature:

25±1 °C

pH:

>= 7.0 at end of test period

Dissolved oxygen:

average minimum oxygen saturation: 58%

Nominal and measured concentrations:

nominal: 8, 16, 32, 64, 128, 256, 512, 1000 mg/L (dilution ratio: 1:2)
measured: deviation < 20%

Details on test conditions:

TEST SYSTEM
- Test vessel: bottles with ground glass stoppers
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 250 mL, no headspace, 250 mL
- Aeration: no (dilution water was oxygen-saturated prior to use)
- Renewal rate of test solution (frequency/flow rate): semistatic conditions (3 times/week)
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 (one additional vessel was set up in order to replace animals without egg packets or very small animals until registration of first offspring)
- No. of vessels per control (replicates): 4 (one additional vessel was set up in order to replace animals without egg packets or very small animals until registration of first offspring)
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: 1 animal / 50 mL

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted freshwater according to German Industrial Standard DIN 38412, Part I and Part II (1982): 11.76 g/l CaCl2*2H2O, 4.93 g/l MgSO4*7H2O, 2.59 g/l NaHCO3, 0.23 g/l KCl
- pH: 8.0±0.2
- Na:K ratio: 10:1
- Culture medium different from test medium: yes


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 9 h light : 15 h dark
- Light intensity: fluorescent light (1.2 to 1.4 W/m²)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- observations: 3 times/week: dead/immobile animals, offspring, pH, dissolved oxygen

VEHICLE CONTROL PERFORMED: not applicable as no vehicle was used

RANGE-FINDING STUDY
- Test concentrations: no data
- Results used to determine the conditions for the definitive study: 24-h EC0 = 1530 mg/L; 24-h EC50 = 2038 mg/L

Reference substance (positive control):

not specified

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks:

of parent animals

Remarks on result:

other: test concentration analytically verified (deviation < 20% from nominal value)

Effect concentrations 2

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

125 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

(reproduction rate)

Remarks on result:

other: test concentration analytically verified (deviation < 20% from nominal value)

Effect concentrations 3

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

250 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

(appearance of first offspring)

Remarks on result:

other: test concentration analytically verified (deviation < 20% from nominal value)

Details on results:

- Mortality of parent animals: 3.3% (9.1% based on 29 control runs)
- No. of offspring produced per day per female: 69 (68; SD = 10.6%; coefficient of variation = 14.8%; based on 29 control solutions)
- Time to first brood release or time to hatch: 7 days (7 days based on 29 control solutions)
- ACR ratio (24-h EC50 = 2038 mg/L) = 127

Results with reference substance (positive control):

- Results with reference substance valid? yes (potassium dichromate)
- Relevant effect levels: 24-h EC50 = 0.93 mg/L (range as stated in OECD TG 202: 0.6 to 2.1 mg/L)

Reported statistics and error estimates:

Student t-test, u-test according to Sachs (1969) as well as graphic determination of dosis-effect curve for parental mortality, reproduction rate and appearance of first offspring

Any other information on results incl. tables

Parental mortality

-      Control after 21 d: 3.3%

-      Highest value not different from control value: 16 mg/L

-      Lowest value different from control value: 32 mg/L (50%)

 

Reproduction rate

-      Number of offspring per animal in control after 21 d: 69

-      Highest value not different from control value: 125 mg/L

-      Lowest value different from control value: 250 mg/L ( 59)

 

First appearance of offspring

-      First day of appearance of offspring in control: 7

-      Highest value not different from control value: 250 mg/L

-      Lowest value different from control value: 500 mg/L (day 9)

 

Chemical analysis

The lowest retrieval was 78% (32 mg/L, aged test solutions, day 7), while the average value is 88% (range 78% to 96%). Therefore it can be concluded that the substance was stable over the exposure period. The measured test concentrations did not deviate by more than 20% from the nominal values, therefore effect values can be based on nominal values.

 

pH values

The dilution water had a pH of 8.0±0.1. The lowest value determined in the test solutions was 7.2. This range is not harmful to Daphnia

 

Dissolved oxygen

The lowest value determined during the 21-d test period was 55% (test solution of 32 mg/L). According to the recent OECD TG 211, the oxygen level should not fall below 60%. Nevertheless, this value can be tolerated by the Daphnia, a negative impact can be excluded on the reproductive behaviour of the test animals regarding the determined effect values.

 

Test solution	Chemical analysis					pH		Dissolved oxygen
(mg/L, nominal)	initial	day 7		day 21		day 7	day 21	day 7	day 21
		fresh	aged (2 d)	fresh	aged (2 d)
0 (control)	not stated
8	not stated
16	not stated					7.5	7.2	64	65
32	28/ 30	28	25	30	27	7.8	7.5	73	70
32*	not stated		26		28	not stated
63	not stated		52		55	not stated
63*	58/ 60		55		57	not stated
125			115		120	not stated
125*	122/ 123		117		118	not stated
250	not stated
500	not stated
1000	not stated
stock solution	1995/ 1996				1994
* blank vessels (without test animals and feed)

 

 

Validity criteria

The validity criteria have been fulfilled.

-      First offspring after < 10 d

-      Reproduction rate > 40

-      Parental mortality <= 20%

Applicant's summary and conclusion

Validity criteria fulfilled:

yes