Reaction products of 2,3-epoxypropyl phenyl ether and 2,2'-iminodi(ethylamine)

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:

February 2018 - May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 2,3-epoxypropyl phenyl ether and 2,2'-iminodi(ethylamine)

EC Number:

948-518-4

Molecular formula:

(C13H23N3O2 . C31H43N3O6)x

IUPAC Name:

Reaction products of 2,3-epoxypropyl phenyl ether and 2,2'-iminodi(ethylamine)

Test material form:

liquid

Details on test material:

PGE-DETA adduct

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.05, 0.16, 0.50, 1.6 and 5 mg/L
- Sampling method: Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).
- Nominal amounts of test item (32 and 100 mg) were each separately added to the surface of 20 liters of culture medium to give the 1.6 and 5.0 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1-Hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 1.6 and 5.0 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions of test item to be present. A series of dilutions was made from the 5.0 and 1.6 mg/L loading rates to give further stock solutions of 0.50, 0.16 and 0.50 mg/L loading rate WAF.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation):
- Method of cultivation:

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not):
- Any deformed or abnormal cells observed:

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24±1°C

pH:

8.0 at test start, 8.7 after 72 h test duration

Nominal and measured concentrations:

0.05, 0.16, 0.50, 1.6 and 5 mg/L nominal Loading Rate

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass conical flasks
- Type: plugged with polyurethane foam bungs
- Aeration: Flasks were constantly shaken at approximately 150 rpm for 72 hours
- Initial cells density: 5.00 x 10E3 cells per mL
- No. of organisms per vessel: 9 flasks

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH:
- Photoperiod:
- Light intensity and quality: white light (380-730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter® Multisizer Particle Counter
- other: At the end of the test, shape and size of the algal cells was inspected microscopically.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limits 0.58 to 0.84 mg/L loading rate WAF

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.097 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

biomass

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.26 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate WAF

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits 0.23 to 0.30 mg/L loading rate WAF

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities detected in the control, 0.050 and 0.16 mg/L loading rate WAFs; misshapen cells or cell debris was observed to be present in the test cultures at 0.50, 1.6 and 5.0 mg/L loading rate WAF.

- Effect concentrations exceeding solubility of substance in test medium:
Observations on the test media were carried out during the mixing and testing of the WAFs.
At both the start and end of the mixing period and following a 1-Hour standing period, the 1.6 mg/L loading rate WAF was observed to have formed a colorless media column with very fine globules of test item dispersed. The 5.0 mg/L loading rate WAF was observed to have formed a colorless media column with large pieces of test item floating at the media surface and settled on the bottom with fine globules of test item dispersed throughout.
After a 1-Hour standing period the 1.6 mg/L loading rate WAF was observed to be a clear colorless media column whilst the 5.0 mg/L loading rate WAF was observed to be a clear colorless media column with test item floating at the media surface and settled on the bottom of the vessel.

Microscopic examination of the WAFs showed there to be no micro-dispersions of test item present.

Results with reference substance (positive control):

- Results with reference substance valid? yes

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results over a 72-Hour period:

ErC50:1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
EyC50: 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L

No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Any other information on results incl. tables

The following data show that the cell concentration of the control cultures increased by a factor of 183 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Nominal cell density of control at 0 hours:       5.00 x10³ cells per ml

Mean cell density of control at 72 hours:         9.16 x10⁵ cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 12% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%. The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hour) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Inhibition of Growth Rate and Yiel in the Definitive Test

Nominal Loading Rate (mg/L)		Growth Rate (cells/ml/hour)		Yield (cells/ml)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.070	-	7.68E+05	-
	R2	0.073		9.81E+05
	R3	0.074		1.06E+06
	R4	0.071		8.33E+05
	R5	0.072		8.84E+05
	R6	0.073		9.39E+05
	Mean	0.072		9.11E+05
	SD	0.001		1.05E+05
0.050	R1	0.071	1	7.97E+05	[4]
	R2	0.075	[4]	1.13E+06
	R3	0.072	0	9.09E+05
	Mean	0.073	[1]	9.45E+05
	SD	0.002		1.68E+05
0.16	R1	0.068	6	6.49E+05	27**
	R2	0.068	6	6.49E+05
	R3	0.069	4	6.99E+05
	Mean	0.068	5	6.66E+05
	SD	0.001		2.85E+04
0.50	R1	0.052	28	2.08E+05	80**
	R2	0.047	35	1.41E+05
	R3	0.052	28	2.03E+05
	Mean	0.050	30**	1.84E+05
	SD	0.003		3.74E+04
1.6	R1	0.018	75	1.30E+04	99**
	R2	0.009	88	4.80E+03
	R3	0.001	99	4.27E+02
	Mean	0.009	87**	6.08E+03
	SD	0.009		6.39E+03
5.0	R1	-0.008	111	-2.15E+03	100**
	R2	-0.007	110	-1.89E+03
	R3	-0.007	110	-2.01E+03
	Mean	-0.007	110**	-2.02E+03
	SD	0.001		1.32E+02

* In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R = Replicate

- = Not applicable

SD = Standard deviation

[ ] = Increase in growth compared to controls

** = Statistically significant from the control

Applicant's summary and conclusion

Conclusions:

Inhibition of Growth Rate:
ErL10 (0 to 72 hour) : 0.29 mg/L loading rate WAF
ErL20 (0 to 72 hour) : 0.40 mg/L loading rate WAF
ErL50 (0 to 72 hour) : 0.70 mg/L loading rate WAF; 95% confidence limits 0.58 to 0.84 mg/L loading rate WAF

Inhibition of Yield:
EyL10 (0 to 72 hour) : 0.097 mg/L loading rate WAF
EyL20 (0 to 72 hour) : 0.14 mg/L loading rate WAF
EyL50 (0 to 72 hour) : 0.26 mg/L loading rate WAF; 95% confidence limits 0.23 to 0.30 mg/L loading rate WAF

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009.

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

Following preliminary range-finding tests and an initial experiment, Pseudokirchneriella subcapitata was exposed to WAFs of the test item over a range of nominal loading rates of 0.050, 0.16, 0.50, 1.6 and 5.0 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

 

Analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.011 mg/L, to 4.8 mg/L. A decline in measured test concentration was observed at 72 hours in the range of less than the LOQ to 3.7 mg/L.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

Response Variable	EL50 (mg/L Loading Rate WAF)	95% Confidence Limits (mg/L Loading Rate WAF)	No Observed Effect Loading Rate (NOEL) (mg/L)	Lowest Observed Effect Loading Rate (LOEL) (mg/L)
Growth Rate	0.07	0.58 - 0.84	0.16.	0.50
Yield	0.26	0.23 - 0.30	0.050	0.16