N-Phenyl-diethanolamine, reaction products with formaldehyde

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 August 2020 - 01 October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with relevant OECD test guideline and GLP. All validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

23 March 2006; Annex 5 corrected 28 July 2011

Deviations:

yes

Remarks:

pH of solutions not measured at end of final test; algae not observed under microscope. Comparable analytical results between combined limit/R-F test indicate general test conditions were likely comparable too. No effect on integrity of study concluded

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples for possible analysis were taken from all test concentrations and the control.
- Sampling method: Samples, with a volume of 1.0 mL from the approximate centre of the test vessels, were taken at t=0 h and t=72 h. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at 10 mg/L but without algae and samples for analysis were taken at the start and at the end of the test period.
- Sample storage conditions before analysis: Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Since the test item was shown to be hydrolytically unstable (see Sec 5.1.2. Hydrolysis record in this data set), the method of preparation was based on the principles laid down in the OECD Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals. In Test Facility Reference No. 20194879, it was confirmed that the half-life time of the test item was below 1 hour in aqueous solutions at pH values relevant for the present study. It was therefore decided, in agreement with OECD GD 23, to test the resulting hydrolysis product. In Test Facility Reference No. 20194899 (see Sec 8 Analytical methods record in this data set), it was additionally confirmed that in aqueous solutions the reaction product of n-phenyl-diethanolamine with a single molecule of formaldehyde is converted back to n-phenyl-diethanolamine.
Based on these results, it was concluded to prepare nominal concentrations of the parent test item in test medium, mix the solution sufficiently long for complete hydrolysis, and to use the resulting solutions in the further tests. Analytical measurements were performed for both components, i.e. the parent test item n-phenyl-diethanolamine, reaction products with formaldehyde and the hydrolysis product n-phenyl-diethanolamine.

The preparation of test solutions started with the highest concentration of 100 mg/L applying a three-hour period of magnetic stirring to ensure maximum hydrolytic conversion of the test item. After the three-hour period of magnetic stirring the lower test concentrations were prepared by subsequent dilutions of the highest test concentration in test medium. All test solutions were clear and colourless at the end of the preparation procedure.
Due to indicated light sensitivity, glassware was wrapped in aluminum foil and preparation of test solutions was performed under dimmed light conditions to minimise exposure to light.
After test solution preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.
- Eluate: N/A
- Differential loading: N/A
- Controls: Test medium without test item or other additives.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Test concentration separation factor: 3.2
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None reported
- Other relevant information: N/A

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21 - 24 °C. Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.


ACCLIMATION
- Acclimation period: N/A
- Culturing media and conditions (same as test or not): The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not reported

Test temperature:

23 ºC

pH:

8.1 - 8.2

Dissolved oxygen:

Not reported

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal: 1.0, 3.2, 10, 32, 100 mg/L; respectively: 0.73, 2.3, 7.0, 23, 71 mg/L measured.
Nominal concentrations were prepared using the parent test item, whereby the solution was mixed for sufficient time to allow for complete hydrolysis. Analytical measurements were performed for both the parent test item n-phenyl-diethanolamine, reaction products with formaldehyde; and the hydrolysis product n-phenyl-diethanolamine.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL flasks
- Type (delete if not applicable): open - perforated for ventilation
- Material, size, headspace, fill volume: All-glass, aluminium caps, containing 50 mL test solution. Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
- Aeration: Continuous for stock culture suspensions only
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): N/A
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 232.31 x 10^4
- No. of organisms per vessel: N/A
- No. of vessels per concentration (replicates): 3 (additional 1 or 2 replicates of each test concentration without algae)
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): N/A

GROWTH MEDIUM
- Standard medium used: yes: M1: stock culture medium formulated under Milli-RO water in accordance with NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”); M2: pre-culture medium formulated using Milli-RO water in accordance with OECD 201 guideline

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap-water purified by reverse osmosis) prepared by Millipore Corp., Bedford, Mass., USA
- Total organic carbon: N/A
- Particulate matter: N/A
- Metals: N/A
- Pesticides: N/A
- Chlorine: N/A
- Alkalinity: N/A
- Ca/mg ratio: N/A
- Conductivity: N/A
- Culture medium different from test medium: Test medium = M2 (pre-culture culture medium)
- Intervals of water quality measurement: pH measured at the beginning of the test (measurement at end of test not performed, in error); temperature measured continuously in temperature control vessel

OTHER TEST CONDITIONS
- Sterile test conditions: Not reported
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: TLD-lamps with a light intensity within the range of 86 to 87 µE.m-2.s-1.
- Salinity (for marine algae): N/A

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank.
- Chlorophyll measurement: N/A
- Other: N/A

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: N/A
Combined limit/range-finding test:
- Test concentrations: The measured concentrations for the hydrolysis product were 0.071, 0.81, 7.1 and 71 mg/L in solutions prepared at nominally 0.10, 1.0, 10 and 100 mg parent/L, respectively, at the start of the test.
6 replicates of exponentially growing algae were exposed to a control and a nominal concentration of 100 mg/L (parent test item). Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Three replicates per concentration were exposed to nominally 0.10, 1.0 and 10 mg/L (parent test item) in the combined/limit range-finding test.
• Cell densities were recorded at 24 h intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.
• One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval.
• pH was only measured in the control and the highest test concentration.
• Samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Samples taken from all test concentrations were analysed. No peaks related to the parent test item could be detected in any of the samples, indicating that the parent had been completely hydrolysed. During the exposure period, the concentrations generally remained stable, i.e. were at 89 - 108 % relative to the initial concentrations at the end of the test. Only the lowest concentration was found to decrease more strongly and was at 37 % relative to the initial concentration at the end of the test.
The concentrations measured in the samples taken from solutions with algae were comparable with the concentrations measured in the samples without algae. It was thus concluded that the presence of the algae did not affect the concentration of the test item in test medium throughout the test.
- Results used to determine the conditions for the definitive study: Dose-related inhibition of algal growth rate and yield was found at test concentrations from nominally 10 mg/L upwards at the end of the test, resulting in 95 % and 100 % inhibition of growth rate and yield, respectively, at the highest concentration.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: n-phenyl-diethanolamine and other potential smaller amounts of the original parent test item

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: n-phenyl-diethanolamine and other potential smaller amounts of the original parent test item

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: n-phenyl-diethanolamine and other potential smaller amounts of the original parent test item

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

23 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: n-phenyl-diethanolamine and other potential smaller amounts of the original parent test item

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): At the end of the final test, no microscopic observations were performed on any test concentration and the control to observe for any abnormal appearance of the algae in error.
- Unusual cell shape: Information not available
- Colour differences: Information not available
- Flocculation: None reported
- Adherence to test vessels: None reported
- Aggregation of algal cells: None reported
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes: The batch of Raphidocelis subcapitata tested, showed expected sensitivity to potassium dichromate based on the historical range of reference tests performed by the Test Facility in the last ten years.
- EC50: Not provided, results available in Test Facility Study No. 20270038 (21-24 September 2020)
- Other: N/A

Reported statistics and error estimates:

For determination of the NOEC and the ECx the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of growth rate (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller) or inhibition of yield (Step-down Jonckheere-Terpstra Test Procedure, α=0.05, one-sided, smaller).
Calculation of ECx-values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding geometric mean concentrations of the hydrolysed test item.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Validity criteria

1. In the control, cell density increased by an average factor of at least 16 within the exposure period (measured as: 232).
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35 % (measured as: 13 %).
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 % (measured as: 2.1 %).

Analysis

Samples taken from all test concentrations were analysed. No peaks related to the parent test item could be detected in any of the samples throughout the study, indicating that the parent had been completely hydrolysed.

The measured concentrations for the hydrolysis product were 0.73, 2.3, 7.0, 23 and 71 mg/L in solutions prepared at nominally 1.0, 3.2, 10, 32 and 100 mg_parent/L, respectively, at the start of the test. During the exposure period, the concentrations remained stable, i.e. were at 91 - 103 % relative to the initial concentrations at the end of the test. Based on these results, the geometric mean measured concentrations of n-phenyl-diethanolamine were calculated and used to express effect parameters (see Table 5).

Table 1. Geometric Mean Measured Versus Nominal Concentrations

n-phenyl-diethanolamine, reaction  products with formaldehyde   Nominal conc. (mg/L)	n-phenyl-diethanolamine Measured conc. (mg/L)		n-phenyl-diethanolamine Geometric mean conc. (mg/L)
	t=0h	t=72h
1.0	0.726	0.685	0.71
3.2	2.25	2.14	2.2
10	7.03	7.04	7.0
10*	6.68	7.04	6.9
32	23.2	21.2	22
100	71.3	73.5	72

* Without algae

Table 2. Growth Rate and Percentage Inhibition for the Total Test Period

n-phenyl-diethanolamine Geometric mean conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.814	0.0383	6
0.71	1.832	0.0230	3	-0.96
2.2	1.829	0.0174	3	-0.82
7.0	1.573	0.0147	3	13*
22	0.720	0.0063	3	60*
72	0.529	0.0387	3	71*

* Effect was statistically significant

Table 3. Yield and Percentage Inhibition for the Total Test Period

n-phenyl-diethanolamine Geometric mean measured conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	231.311	25.0771	6
0.71	242.849	17.1135	3	-5.0
2.2	240.874	12.4659	3	-4.1
7.0	111.261	4.9794	3	52*
22	7.675	0.1642	3	97*
72	3.914	0.5570	3	98*

*Effect was statistically significant

Table 4. Growth Rate and Percentage Inhibition at Different Time Intervals

n-phenyl-diethanolamine Geometric mean measured conc. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	2.087		1.710		1.646
0.71	3	2.099	-0.59	1.750	-2.3	1.646	0.024
2.2	3	2.119	-1.5	1.681	1.7	1.687	-2.5
7.0	3	1.885	9.7	1.385	19	1.450	12
22	3	1.053	50	0.434	75	0.674	59
72	3	0.000	100	0.000	100	1.588	3.6

Table 5. Effect parameters

Parameter (mg/L)		NOEC	EC10	EC20	EC50
Growth rate	Value	2.2	3.9	7.2	23
	lower 95%-cl		3.7	6.9	22
	upper 95%-cl		4.2	7.5	24
Yield	Value	2.2	3.4	4.3	6.9
	lower 95%-cl		2.5	3.5	6.5
	upper 95%-cl		4.0	4.8	7.2

Table 6. pH Levels Recorded during the Final Test

n-phenyl-diethanolamine Geometric mean conc. (mg/L)	pH
	t=0h	t=72h
Control	8.2	n.d.
0.71	8.2	n.d.
2.2	8.1	n.d.
7.0	8.1	n.d.
22	8.1	n.d.
72	8.1	n.d.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study with Raphidocelis subcapitata, the test item and other potential smaller amounts of the original parent test item inhibited growth rate and yield of this freshwater algae species significantly at geometric mean concentrations of 7.0 mg/L and higher.
The 72h-ErC50 was 23 mg/L with a 95% confidence interval 22 - 24 mg/L.
The 72h-EyC50 was 6.9 mg/L with a 95% confidence interval 6.5 - 7.2 mg/L.
The 72h-NOEC for growth rate inhibition was 2.2 mg/L based on statistical significance.
The 72h-NOEC for yield inhibition was 2.2 mg/L based on statistical significance.

Executive summary:

A test was performed in accordance with OECD 201, in order to determine the potential of the test item to generate toxic effects in Raphidocelis subcapitata.

The algae was exposed to the test item for 72 h, to determine the NOEC, EC₁₀, EC₂₀ and EC₅₀ for both inhibition of growth rate and inhibition of yield.

A full test was performed based on the results of a preceding combined limit/range-finding test. In the range-finder test, the algae was exposed to nominal concentrations of 0.10, 1.0, 10 and 100 mg/L (0.071, 0.81, 7.1 and 71 mg/L measured). A blank control and 3 replicates per test concentraton were run during the test.

Analytical measurements were performed for both components, i.e. the parent test item n-phenyl-diethanolamine, reaction products with formaldehyde and the hydrolysis product n-phenyl-diethanolamine. Nominal test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (parent test item); corresponding to measured concentrations of 0.71, 2.2, 7.0, 22 and 72 mg/L were utilised in the full test. Samples taken from all test concentrations were analysed. No peaks related to the parent test item could be detected in any of the samples throughout the study, indicating that the parent had been completely hydrolysed. During the exposure period, the concentrations remained stable, at 91 - 103 % relative to the initial concentrations at the end of the test.

The study met the validity criteria prescribed by the study plan and was considered valid. Under the conditions of this study, the test item and other potential smaller amounts of the original parent test item inhibited growth rate and yield of this freshwater algae species significantly at geometric mean concentrations of 7.0 mg/L and higher.

The 72h-ErC50 was 23 mg/L with a 95 % confidence interval 22 - 24 mg/L.
The 72h-EyC50 was 6.9 mg/L with a 95 % confidence interval 6.5 - 7.2 mg/L.
The 72h-NOEC for growth rate inhibition was 2.2 mg/L based on statistical significance.
The 72h-NOEC for yield inhibition was 2.2 mg/L based on statistical significance.

Description of key information

OECD 201, Augusiak (2020), 72 h NOEC: 2.2 mg/L; 72 h ErC10: 3.9 mg/L; 72 h ErC50: 23 mg/L (geo. mean meas.)

Key value for chemical safety assessment

EC50 for freshwater algae:

23 mg/L

EC10 or NOEC for freshwater algae:

2.2 mg/L