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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see "General Justification for Read-Across" attached to IUCLID section 13

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Mutual read across from the AAPBs to one another is justified:

a) Based on the information given in section 1, it can be concluded that all AAPBs mentioned above are similar in structure, since they are manufactured from similar resp. identical precursors under similar conditions and all contain the same functional groups. Thus a common mode of action can be assumed.
b) The content of minor constituents in all products are comparable and differ to an irrelevant amount.
c) The only deviation within this group of substances is a minor variety in their fatty acid moiety, which is not expected to have a relevant impact on intrinsic toxic or ecotoxic activity and environmental fate. Potential minor impact on specific endpoints will be discussed in the specific endpoint sections.

The read-across hypothesis is based on structural similarity of target and source substances. Based on the available experimental data, including key physico-chemical properties and data from toxicokinetic, acute toxicity, irritation, sensitisation, genotoxicity and repeated dose toxicity studies, the read-across strategy is supported by a quite similar toxicological profile of all five substances.
The respective data are summarised in the data matrix; robust study summaries are included in the Technical Dossier in the respective sections.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see "General Justification for Read-Across" attached to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
see "General Justification for Read-Across" attached to IUCLID section 13

4. DATA MATRIX
see "General Justification for Read-Across" attached to IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Key result
Duration:
48 h
Dose descriptor:
EC50
Remarks:
freshwater invertebrates
Effect conc.:
ca. 1.9 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 1.37 - 2.69 mg a.i./L
Key result
Duration:
48 h
Dose descriptor:
LC50
Remarks:
marine invertebrates
Effect conc.:
7 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95% c.i.: 5.4 - 8.3 mg a.i./L
Conclusions:
For freshwater invertebrates (Daphnia magna) the lowest 48 h EC50 was determined to be 1.9 mg a. i./L (nominal), and for the marine copepod Acartia tonsa  the 48 h EC50 was 7.0 mg a.i./L (nominal).
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 18, 2020 - February 20, 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Chemical name (IUPAC): 1-Propanaminium, N-(carboxymethyl)-N,N-dimethyl-3-[(1-oxododecyl)amino]-, inner salt
Analytical monitoring:
yes
Details on sampling:
samples were taken at start and at end after 24 h of exposure the fresh and aged solutions
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
A stock solution of the test item was prepared by dissolving 136.00 mg of the test item at test start and 136.23 mg of the test item after 24 h of exposure in 500 mL of test medium. The resulting stock solutions had a nominal concentration of 272 mg test item/L. These stock solutions were stirred on a magnetic stirrer for 15 and 30 minutes at ambient temperature in the dark. The stock solutions were visually examined for undissolved/particulate matter. The stock solutions were clear, no undissolved matter was observed. The stock solutions were used immediately to prepare the desired test item concentrations.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: Straus , clone M10
- Age at study initiation (mean and range, SD): Less than 24 hours
- Source: inhouse culture; the organisms were originally supplied by KU Leuven, Belgium.
- Age of parental stock (mean and range, SD): Less than 35 days
- Feeding during test : no

ACCLIMATION
- Acclimation conditions (same as test or not): same
- Type and amount of food: Algae (Desmodesmus subspicatus), instant baker’s yeast suspension, TetraMin® suspension
- Feeding frequency: 4 times per week

The selected holding vessels designated for production of the neonates for the test were cleared of all mixed-aged young daphnids on the day before test start. These vessels contained parental daphnids aged less than 35 days. On the day of test start (day 0) all newly produced neonates of these vessels were < 24 hours old. These neonates were collected for the test.

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
19.5 - 21.3°C
pH:
7.8 - 7.9
Dissolved oxygen:
8.5 - 9.4mg/L (96 - 102% of air saturation value)
Nominal and measured concentrations:
nominal: 11.6,
25.5, 56.2, 124 and 272 mg test item/L, which correspond to the following concentrations of 4.27, 9.39, 20.7, 45.5 and 100 mg solids/ L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass vessels (nominal volume: 100 mL), covered with watch glasses, fill volume: 50–60 mL
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): Semi static system; renewal after 24 h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
Medium M4 (OECD 2004)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light: 8 hours dark
- Light intensity: 50–1000 lx

EFFECT PARAMETERS MEASURED:
Immobility, 24h, 48 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
124 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
45.6 other: mg solids/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Other biological observations: After 24h daphnids showed reduced swimming activity in the C4 treatment and lied on the bottom of the test vessels in the highest treatment. After 48h the daphnids of the C4 treatment showed reduced swimming activity and lied on the bottom of the test vessels. In addition, one daphnid lied on the bottom of a vessel in the C2 concentration level. No sublethal observations on the mobile daphnids were observed in the other treatment levels and the control.
- Mortality of control: no
Results with reference substance (positive control):
- Results with reference substance valid?
- Relevant effect levels: EC50(24 h) = 1.484 mg/L (valid; According to the OECD guideline No. 202 (OECD, April 2004) EC50(24 h)-values for potassium dichromate obtained from different laboratories should range between 0.6 mg/L and 2.1 mg/L)
- tested November 2019
Reported statistics and error estimates:
Mortality was evaluated as assessed by immobility of the daphnids. Probit analysis using linear maximum likelihood regression was used to estimate the ECx values.
The statistical software package ToxRat 3.3 Professional (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf) was used for these calculations.

All validity criteria were fulfilled as required by the test guideline:

Required: immobile daphnids in the control:</=10% (this study: 0%)

Required: dissolved oxygen concentration in control and test vessels at the end of the test:>/=3 mg/L (this study:>/=8.5 mg/L)

Signs of disease or stress, for example, discoloration or unusual behaviour, such as being trapped at the water surface, did not exceed 10 per cent of the daphnids in the controls.

Analytical results

The test solutions, 11.6, 25.5, 56.2, 124 and 272 mg test item/L, which correspond to the following concentrations of 4.27, 9.39, 20.7, 45.5 and 100 mg solids/ L. were analysed to

determine actual levels of the test item at start and at end of the test. Analysis of the test item in the samples was performed using an HPLC-MS/MS method.

The limit of quantification (LOQ) was 5 mg test item/L.

Analysis of control samples of test medium yielded no residues at or above 30% of the limit of quantification.

The measured concentrations ranged between 112117% of the nominal concentrations at the middle and highest treatment level throughout the test. No biological effects were observed at the three lower concentrations. The lowest test concentration showed similar ranges from 121126% of the nominal concentrations but was slightly higher than the required 120% limit of the nominal concentration throughout the test period.

Since the measured test item concentrations were stable during the test period between 98 and 101% of the measured initial concentrations and the overall recovery of the measured concentrations was 118% of the nominal concentrations, the biological results are calculated and reported based on nominal concentrations.

Introduced number (0 h), total number of immobile (24 and 48 h) Daphnia magna dependent on test concentration and time.

 

Nominal concentration

[mg test item/L]

 

Replicate

Control

11.6

25.5

56.2

124

272

 

0 h

(introduced)

A

5

5

5

5

5

5

B

5

5

5

5

5

5

C

5

5

5

5

5

5

D

5

5

5

5

5

5

Sum introduced:

 

20

20

20

20

20

20

24 h

immobile

A

0

0

0

0

0

2

B

0

0

0

0

0

1

C

0

0

0

0

0

0

D

0

0

0

0

0

0

Sum immobile:

 

0

0

0

0

0

3

48 h

immobile

A

0

0

0

0

0

5

B

0

0

0

0

2

5

C

0

0

0

0

3

5

D

0

0

1

0

3

5

Sum immobile:

 

0

0

1

0

8

20

 

 

Observations concerning the behaviour of the mobile daphnids (number of daphnids showing the effect described).

 

Nominal concentration

[mg test item/L]

 

Replicate

Control

11.6

25.5

56.2

124

272

 

introduced:

 

20

20

20

20

20

20

24 h

 

A

0

0

0

0

5c

3b

B

0

0

0

0

5c

4b

C

0

0

0

0

5c

5b

D

0

0

0

0

5c

5b

Mobile

 

20

20

20

20

20

17

48 h

 

A

0

0

0

0

5c

-

B

0

0

0

0

3c

-

C

0

0

0

0

2c

-

D

0

0

1b

0

1b

-

Mobile

 

20

20

19

20

12

0

 

0 No adverse observations compared to the controls; no special observations in the controls.

b Daphnids were lying on the vessel bottom

c Daphnids showed reduced swimming activity compared to the controls

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of C12 AAPB to Daphnia magna was investigated in a study conducted according to OECD Guideline 202. A 48 h EC50= 124 mg/L as test material, corresponding to 45.6 mg solids/L was determined.
Executive summary:

A study on the acute toxicity of C12 AAPB to Daphnia magna was conducted according to OECD Guideline 202 (April 2004).

The daphnids (20/concentration) were exposed to nominal concentrations of 0 (control),11.6, 25.5, 56.2, 124 and 272 mg test item/L, which correspond to the following concentrations of 4.27, 9.39, 20.7, 45.5 and 100 mg solids/ L, for 48 h under semi-static conditions.

The measured concentrations ranged between 112117% of the nominal concentrations at the middle and highest treatment level throughout the test. No biological effects were observed at the three lower concentrations. The lowest test concentration showed similar ranges from 121126% of the nominal concentrations but was slightly higher than the required 120% limit of the nominal concentration throughout the test period.

Since the measured test item concentrations were stable during the test period between 98 and 101% of the measured initial concentrations and the overall recovery of the measured concentrations was 118% of the nominal concentrations, the biological results are calculated and reported based on nominal concentrations.

A clear concentration-response relationship was observed. The following EC50 value was determined:

48 h EC50 = 124 mg test item/L, corresponding to 45.6 mg solids/L

Description of key information

The acute toxicity of C12 AAPB, C8 -18 and C 18 unsatd. AAPB to Daphnia magna was investigated in four studies conducted according to OECD guideline 202. The study with the lowest 48 h EC50 was selected as key study. Additional data were available for C8-18 AAPB from an OECD 202 guideline study with Daphnia magna. The acute toxicity of Coco AAPB to the marine copepod Acartia tonsa was investigated in a study conducted according to ISO 14669 (1999) Water 
Quality - Determination of the acute lethal toxicity to marine capepods (Copepoda; Crustacea).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
1.9 mg/L

Marine water invertebrates

Marine water invertebrates
Effect concentration:
7 mg/L

Additional information

Reliable results on the acute toxicity to fresh water invertebrates obtained in guideline studies are available for C12 AAPB and the closely related source substances C8-18 AAPB C8-18 and C18 unsatd. AAPB, as well as data on the acute toxicity of C8-18 and C18 unsatd. AAPB to salt water invertebrates

A justification for read-across is given below.

 

Freshwater invertebrates

The acute toxicity of Coco AAPB toDaphnia magnawas investigated in a study conducted according to OECD TG 202 / part I (1984) under static conditions.Daphnia magnawas exposed to 6 nominal concentrations ranging from 0.5 to 16 mg product/L. A 48 h LC50 of ca. 1.9 mg a.i/L (nominal) was determined based on an active matter content of 30%.

 

The 48 hr-acute toxicity of C8-18 AAPB (30% active matter) toDaphnia magnawas investigated in a study conducted according to OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test) and EU Method C.2 (Acute Toxicity for Daphnia) under static conditions. Daphnids were exposed to control and test chemical at nominal concentrations of 6.25; 12.5; 25; 50; 100 mg product/L for 48 hr. The test material concentrations were analysed via UV absorption at 485 nm. The C8-18 AAPB concentrations were >80% (exception: 72.2% at the start in the 6.25 mg product/L group). The 48 h EC50 was determined to be 6.5 mg a.i./L based on nominal concentrations.

 

A study on the acute toxicity of C12 AAPB to Daphnia magna was conducted according to OECD Guideline 202 (April 2004). The daphnids (20/concentration) were exposed to nominal concentrations of 0 (control),11.6, 25.5, 56.2, 124 and 272 mg test item/L, which correspond to the following concentrations of 4.27, 9.39, 20.7, 45.5 and 100 mg solids/ L, for 48 h under semi-static conditions.

The measured concentrations ranged between 112–117% of the nominal concentrations at the middle and highest treatment level throughout the test. No biological effects were observed at the three lower concentrations. The lowest test concentration showed similar ranges from 121–126% of the nominal concentrations but was slightly higher than the required 120% limit of the nominal concentration throughout the test period.

Since the measured test item concentrations were stable during the test period between 98 and 101% of the measured initial concentrations and the overall recovery of the measured concentrations was 118% of the nominal concentrations, the biological results are calculated and reported based on nominal concentrations.

A clear concentration-response relationship was observed. The following EC50 value was determined:

48 h EC50 = 124 mg test item/L, corresponding to 45.6 mg solids/L

 

Two further supporting studies are available:

The acute toxicity of Coco AAPB to Daphnia magna was investigated in a study conducted according to OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test) and EU Method C.2 (Acute Toxicity for Daphnia). The daphnids were exposed to nominal concentrations of 0 (control), 0.56, 1.0, 1.8, 3.2, 5.6, and 10.0 mg a.i./L for 48 h under semi-static conditions (renewal of the test solutions after 24 h). The 48 h EC50 was found to be 4.1 mg a.i./L (nominal).

 

The 48-hr-acute toxicity of Coco AAPB to Daphnia magna was investigated in a study conducted according to EU Method C.2 (Acute Toxicity for Daphnia) under static conditions. Daphnids were exposed to control and test chemical at (nominal/measured) concentration of 3.5; 6; 10; 17; 30 mg dry residue/L for 48 hr. Immobilization were observed. The 48 h EC50 was 6.6 mg a.i./L nominal.

 

Marine invertebrates

The acute toxicity of Coco AAPB to the marine copepod Acartia tonsa was investigated in a study conducted according to ISO 14669 (1999) Water Quality - Determination of the acute lethal toxicity to marine capepods (Copepoda; Crustacea). Acartia tonsa were exposed to nominal Coco AAPB static conditions for 48 h. The 48 h NOEC and LC50 values were determined to be 3.6 mg a. i./L and 7.0 mg a.i./L nominal, respectively. The 48 h LC50 of 7.0 mg a i./L nominal was used for further assessment.

 

Conclusion

For freshwater invertebrates (Daphnia magna) the lowest 48 h EC50 was determined to be 1.9 mg a. i./L (nominal), and for the marine copepod Acartia tonsa  the 48 h EC50 was 7.0 mg a.i./L (nominal).

 

 

Justification for read-across

For details on substance identity and detailed (eco)toxicological profiles, please refer also to the general justification for read-across given at the beginning of the CSR and attached as pdf document to IUCLID section 13.

 

This read-across approach is justified based on structural similarities. All AAPBs contain the same functional groups. Thus a common mode of action can be assumed.

The only deviation within this group of substances is a minor variety in their fatty acid moiety (chain length and degree of unsaturation), which is not expected to have a relevant impact on intrinsic ecotoxicological properties.

 

a. Structural similarity and functional groups

Alkylamidopropyl betaines (AAPBs) are – with the exception of C12 AAPB - UVCB substances (Substances of Unknown or Variable composition, Complex reaction products or Biological materials), which are defined as reaction products of natural fatty acids or oils with dimethylaminopropylamine and further reaction with sodium monochloroacetate. AAPBs are amphoteric surfactants, which are characterized by both acidic and alkaline properties.

 

Their general structure is:

 

R-C(O)-NH-(CH2)3-(N(CH3)2)+-CH2-C(O)O-

R = fatty acid moiety

 

The fatty acids have a mixed, slightly varying composition with an even numbered chain length from C8 to C18. Unsaturated C18 may be included. Consequently, the AAPBs differ by their carbon chain length distribution and the degree of unsaturation in the fatty acid moiety. However, Lauramidopropyl betaine (C12 fatty acid derivate) is the major ingredient of all AAPBs covered by this justification as listed in table 1 “Substance identities” of the general justification for read-across.

 

The substances under evaluation share structural similarities with common functional groups (quaternary amines, amide bonds and carboxymethyl groups), and fatty acid chains with differences in chain length and degree of saturation.

 

b. Differences

Differences in acute ecotoxicity of the AAPBs could potentially arise from the following facts:

-Different amounts of different carbon chain lengths (carbon chain length distribution):

Higher amounts of higher chain lengths and corresponding lower amounts of lower chain length could result in a rising average lipophilicity. However, the main component for all AAPBs is C12 AAPB. Relevant effects on ecotoxicity are not to be expected.

- Different amounts of unsaturated fatty ester moieties:

Effects may be expected for e.g. physical state, but are not considered to be of relevance for ecotoxicity.

 

Comparison of short-term invertebrate toxicity data

 

Endpoints

Source substances

Target substance

 

C8-18 AAPB

C8-18 and C18 unsatd. AAPB

C12 AAPB

Short-term toxicity to aquatic invertebrates

key_Short-term toxicity to aquatic invertebrates: 97862-59-4_9.1.1_THG_1991c_OECD 202

 

key study

 

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Daphnia magna, static, freshwater

 

48 h EC50 = 6.5 mg/Lact. ingr. (nominal) based on: mobility (95% C.I.: 4.8 - 8.4 mg a.i./L)


24 h EC50 > 30 mg/L
act. ingr. (nominal) based on: mobility

 

Reliability: 1 (reliable without restriction), GLP

key.Short-term toxicity to aquatic invertebrates: 61789-40-0_9.1.1_KAO Corporation_1992_OECD 202

 

key study

 

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test) (part I, 24h EC50 Acute Immobilization Test (1984))


Daphnia magna, static, freshwater

 

48 h EC50 ca. 1.9 mg/Lact. ingr. (nominal) based on: mobility (95% CL: 1.37 - 2.69 mg a.i./L)

 

Reliability: 2 (reliable with restrictions), GLP

 

key_Short-term toxicity to aquatic invertebrates: 4292-10-8_9.1.1_Solvay_2020_OECD202

 

key study

 

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Daphnia magna, semi-static, freshwater

 

48 h EC50 = 124 mg/L as test material, corresponding to 45.6 mg solids/L

 

Reliability: 1 (reliable without restriction), GLP

 

key (marine).Short-term toxicity to aquatic invertebrates.61789-40-0_9.1.1__Marine_48h_AcartiaTonsa_Rhodia_Mirataine-BET-C37_ISO_2008

 

key study

 

ISO 14669 (1999) Water Quality - Determination of the acute lethal toxicity to marine copepods (Copepoda; Crustacea)


Acartia tonsa, static,
saltwater


48 h NOEC = 10 mg/L
test mat. (nominal) based on: mobility (95% c.i.: 14.85-23.01 mg/L)


48 h NOEC = 3.6 mg/L
act. ingr. (nominal) based on: mobility (95% c.i.: 5.4 - 8.3 mg a.i./L)

 

48 h LC50 = 19.38 mg/Ltest mat. (nominal) based on: mobility (95% c.i.: 14.85-23.01 mg/L)


48 h LC50 = 7 mg/L
 act. ingr. (nominal) based on: mobility (95% c.i.: 5.4 - 8.3 mg a.i./L)

 

Reliability: 1 (reliable without restriction), GLP

 

sup.Short-term toxicity to aquatic invertebrates.61789-40-0_9.1.1_Acute_Daphnia_Unilever_A17-AT-L17-01

supporting study


OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

 

Daphnia magna, semi-static, freshwater

 
48 h EC50 = 4.1 mg/L
act. ingr. (nominal) based on: mobility (95% c.i.: 3.3-5.3 mg a.i./L)

 

Reliability: 2 (reliable with restrictions), GLP

 

sup.Short-term toxicity to aquatic invertebrates: 61789-40-0_9.1.1_Hüls_1996_EEC 92-69 C2

 

supporting study

 

EU Method C.2 (Acute Toxicity for Daphnia)


Daphnia magna, static, freshwater

 

48 h EC0 < 3 mg/Lact. ingr. (nominal) based on: mobility
48 h EC100 > 25 mg/L
act. ingr. (nominal) based on: mobility
48 h EC50 = 6.6 mg/L
act. ingr. (nominal) based on: mobility (95% c.i. 4 - 11 mg/L)

 

Reliability: 2 (reliable with restrictions), GLP

 

The 48 h EC50 values of C8-18 AAPB and C8-18, C18 unsatd. AAPB to Daphnia magnawere in the range of 1.9 – 6.6 mg a.i./L. The sensitivity of the marine invertebrateAcartia tonsa was in the same order of magnitude (48 h EC50 = 7 mg a.i./L)

 

 

Quality of the experimental data of the analogues:

The available data are adequate and sufficiently reliable to justify the read-across approach.

The studies withDaphnia magnawere conducted according toOECD Guideline 202orEU Method C.2 and were reliable or reliable with restrictions (RL1-2, GLP).

A further study with the saltwater organismAcartia tonsawas conducted according toISO 14669(RL1, GLP)

The test materials used in the respective studies represent the source substance as described in the hypothesis in terms of substance identity and minor constituents.

Overall, the study results are adequate for the purpose of classification and labelling and risk assessment.

 

Conclusion

Based on structural similarities of the target and source substancesas presented above and in more detail in the general justification for read across, it can be concluded that the available data from the source substancesC8-18 AAPB and C8-18 and C18 unsatd. AAPB are also valid for the target substance C12 AAPB.

 

For freshwater invertebrates (Daphnia magna) the lowest 48 h EC50 was determined to be 1.9 mg a. i./L (nominal), and for the marine copepod Acartia tonsa the 48 h EC50 was 7.0 mg a.i./L (nominal).