4-((3-acetoxypropyl)amino)-2,2-dimethyl-4-oxobutane-1,3-diyl diacetate

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:

29 Aug 2017 - 17 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2011

Deviations:

yes

Remarks:

Only the highest test concentration was analysed and only one sample of the stock solution taken at start of the exposure period. The deviations from the guideline have no influence on the integrity of this study (see "any other information on results").

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-(acetyloxy)-1-{[3-(acetyloxy)propyl]carbamoyl}-2,2-dimethylpropyl acetate

Cas Number:

98133-47-2

Molecular formula:

C15H25NO7

IUPAC Name:

3-(acetyloxy)-1-{[3-(acetyloxy)propyl]carbamoyl}-2,2-dimethylpropyl acetate

Test material form:

liquid: viscous

Specific details on test material used for the study:

Batch No.: 75518
Purity: 95.7%
Expiration Date: September 19, 2018

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples of stock solution and test solutions were taken to determine the actual test item concentrations in comparison to the nominally applied concentrations.
Samples from the test solutions were analysed at start of exposure period (0 hours) and at the end of the exposure (72 hours).

Test solutions

Vehicle:

no

Details on test solutions:

Based on the results of a preliminary non-GLP range finding test, the following concentration steps in a geometrical series (spacing factor: 2.0) were tested in the definitive test: 25.0, 50.0, 100, 200 and 400 mg test item/L.
A stock solution was prepared by directly adding 0.8054 g test item in 2000 ml of growth medium, resulting in a nominal loading rate of 400 mg test item/L. This stock solution (S1) was stirred at 500 rpm using a magnetic stirrer for 13 min at ambient temperature in the dark. Thereafter this stock solution was left to settle for 20 minutes. The stock solution was visually examined for undissolved/particulate matter in the free water column. No undissolved/particulate matter was observed and therefore S1 was used for preparation of test solutions.
After preparation the test solutions were stirred for ca. 10 minutes on a magnetic stirrer at room temperature and placed into a temperature controlled room for temperature adaptation.
After temperature adaptation of the test solutions, 1.577 or 0.788 mL of the algal pre-culture (Pseudokirchneriella subcapitata) were added to each of the volumetric flasks containing 1000 or 500 mL of the test solutions, respectively to achieve an algal cell concentration of approximately 0.5x 104 cells/mL. These manipulations were performed under a laminar flow box. The test solutions were homogenised by manual shaking 15 times overhead, before adding 100±5 mL of the test media to each test vessel to ensure a homogeneous distribution of the algal cells.

Test organisms

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata (SAG 61.81), currently valid species name: Raphidocelis subcapitata
Suppier: Sammlung von Algenkulturen, Albrecht-von-Haller-Institut,Universität Göttingen, Germany
Date of receipt: February 24, 2016
Acclimation: To adapt the algae to the test conditions a pre-culture was inoculated by a liquid algal culture and incubated under test conditions.
Age of pre-culture (at test initiation): 4 days

Study design

Test type:

static

Water media type:

freshwater

Remarks:

Mod OECD medium (OECD 201, EN ISO 8692)

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.0-22.5, mean 22.2 °C

pH:

Of test media:7.9
In test solutions: 7.8-9.1

Nominal and measured concentrations:

Nominal: 25.0, 50.0, 100, 200 and 400 mg test item/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass Erlenmeyer flasks (300 mL) covered by air-permeable lids
- Volume of test solution per test vessel: 100±5 mL
- Initial cells density: 0.5x104 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: OECD medium (modified)
- Prepared with a 10-fold concentrated iron (Fe)-EDTA complex compared to the original recipe according to guideline OECD 201
- Justification for modification: The steady and sufficient supply of trace elements (e.g. iron) is essential for algal growth. In this context, the introduction of chelating agents (e.g. EDTA) is very valuable, since by using these compounds, trace element cations easily form reversible complexes. The splitting of these complexes steadily supplies the algae with the amounts of trace elements required for growth. There was no indication of interaction between this medium and the test item.

OTHER TEST CONDITIONS
- Sterile test conditions: yes; The algal medium was sterilised (filtered, pore size 0.2 pm) before use. The vessels used were sterilised using a drying oven for three hours at 150 °C.
- Adjustment of pH: Before use medium adjusted to 8.1±0.2
- Photoperiod:24 h light/0 h dark
- Light intensity:63.2-70.0 pE M-2S-1; mean 67.8 pE m-2s-1;
- Type of Light: Econlux LED Sunstrip "daylight"

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- At start of the test, the cell numbers were determined microscopically using a counting chamber (Thoma chamber). Occurrence of morphological deviations of cells was checked.
- After 24, 48 and 72 hours, the cell numbers were determined by measuring the fluorescence intensity in 4 samples of 200 pL of test solution per replicate using a fluorometer (Multiple Reader Tecan ULTRA).
- At the end of exposure, morphological deviations compared to the control cells were recorded.

Reference substance (positive control):

yes

Remarks:

A reference test using potassium dichromate (K2Cr207) was performed in April 2017 in a separate study (study number: IA01704).

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 400 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 400 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Details on results:

Analytical findings:
The highest concentration was analysed to determine actual levels of the test item at start of exposure period (time 0 hours) and at the end of the exposure at 72 hours. Since no biological effects were observed no further concentration levels were analysed. The recoveries were determined to be 83-96 % of the nominal concentration and 87 % initial measured concentration. Thus, the test item concentration based on nominal and initial measured concentration remained stable within ±20% throughout the exposure period. Therefore, the biological results are calculated and reported based on nominal concentrations at each concentration level.

Biologcal findings:
In none of the concentration levels (control and C1-C5, up to 400 mg test item/L), deformed and/or damaged algal cells were observed during microscopic inspection.
No concentration-response relationship was observed for the biological parameters yield and growth rate during the exposure period.
The EC50 of Pseudokirchneriella subcapitata for yield and growth rate were not statistically determined, since no meaningful inhibition was determined up to the highest tested concentration level. The EC50 of Pseudokirchneriella subcapitata was stated to be >400 mg test item/L based on nominal concentrations.
The NOEC based on nominal concentration appeared to be => 400 mg test item/L for both biological parameters yield and growth rate.

Results with reference substance (positive control):

Growth rate EC50 (0-72 h): 0.860 mg/L (0.836 - 0.885 mg/L; 95%-CL)
This value is within the historical range of the general reference test results of the test laboratory (mean ErC50 value = 1.072 ± 0.357 mg/L) and within the narrow upper (1.433 mg/L) and lower (0.718 mg/L) warning limits.
Furthermore, the ErC50 value is within the refined range of 1.10 ± 0.48 mg/L, recommended based on an international ring test (Pattard, M.; Römbke, J.; Moser, T. (2009).Range of Reference Tests in Aquatic Tests. [in] Moser, H.; Römbke, J. "Ecotoxicological Characterization of Waste. Results and Experiences of an International Ring Test", chapter 5, pp. 61-70.). Therefore the results of this reference test are acceptable and the test conditions are reliable.

Reported statistics and error estimates:

See section 23 of study report

Any other information on results incl. tables

Deviations from the Study Plan and/or the Guideline:

Deviating from the guideline only the highest test concentration was analysed. Since no biological effect was observed in comparison to the control group (all effects levels were below 5 % compared to the control), and the circumstance that the measured concentration based on nominal and initial measured concentration remained stable within ±20% throughout the exposure period, this deviation from the guideline has no influence on the integrity of this study.

Deviating from the study plan only one sample of the stock solution was taken at start of the exposure period. Since the nominal concentration of the stock solution was identical to the highest concentration level and the circumstance that the measured concentration of the highest concentration level based on nominal and initial measured concentration remained stable within ±20% throughout the exposure period, this deviation from the guideline has no influence on the integrity of this study.

Validity Criteria:

The following criteria were fulfilled as required by the study plan and the guideline:

Required:	Found:
Mean biomass increase in the control cultures: factor of at least 16 within the 72-hour test period	251.0
Mean coefficient of variation for section-by-section specific growth rates in the control cultures: up to 35%	14.7%
Coefficient of variation of average specific growth rates during test period in replicate control cultures: up to 7%	1.1%

The study is therefore considered to be valid.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see "Any other information on results incl Tables"

Conclusions:

In a study to determine the toxicity of the D-Panthenyltriacetate towards the green alga, Pseudokirchneriella subcapitata, no concentration-response relationship was observed for the biological parameters yield and growth rate during the 72 h exposure period.
Since no biological effect was observed in comparison to the control group only the highest test concentration was analysed. The test item was stable, with 83-96 % of the nominal concentration and 87 % of initially measured concentration found in the aged solution. Therefore, the biological results are calculated and reported based on nominal concentrations.
The EC50 of Pseudokirchneriella subcapitata was stated to be > 400 mg test item/L based on nominal concentrations and the NOEC based on nominal concentration appeared to be => 400 mg test item/L for both biological parameters yield and growth rate.