Correction

This article refers to:

Methane reduction, health and regulatory considerations regarding Asparagopsis and bromoform for ruminants

Article title: Methane reduction, health and regulatory considerations regarding Asparagopsis and bromoform for ruminants

Authors: Eason CT, Fennessy P

Journal: New Zealand Journal of Agricultural Research

DOI: http://dx.doi.org/10.1080/00288233.2023.2248948

 

Corrections or additions have been made in three areas.

 

Methane reduction and dose response P.4 to 8:

 

We have recalculated the methane reduction. The analysis of the response to bromoform has been corrected to deal with the conversion from organic matter to dry matter in the complete diet including the Asparagopsis supplement. The study by Li et al. (2018) in sheep has been deleted as the bromoform content of the Asparagopsis as fed is not defined. The data from the study by Alvarez-Hess et al (2023) which was included in the original Table 1 has now been included in the analysis of the mean response and of the regression relationship. Therefore, both Equations 1 and 2 and Figure 1 have been revised.

Table 1. Summary of studies in ruminants fed Asparagopsis^a with details of the studies, and the methane response expressed per mg of bromoform.

Reference and pertinent experimental design details	DMI (kg/day)	Methane (g per kg Dry Matter Intake, DMI)	Asparagopsis inclusion rate in diet (DM basis)	Treatment (days)	Number of animals	Methane response (g/kg DMI compared with control)	Methane response: kg per gram bromoform	Inclusion rate of bromoform (mg/kg diet DM or g/tonne diet DM)	Bromoform (mg per day)	Bromoform (mg per kg LW)
	Dried Asparagopsis systems
Kinley^b	Feedlot steers		Brahman-Angus (477/604 kg)		A. armata – Bromoform (6.55 mg per g seaweed DM)
	9.3	0.2	0.33%	30 + 60	5	10.8	0.50	21.8	202	0.37
	10.8	6.8	0.17%	30 + 60	5	4.2	0.38	10.9	118	0.22
	8.0	10.0	0.08%	30 + 60	5	1.0	0.18	5.4	44	0.08
	8.9	11.0	0.00%	30 + 60	5	0	0	0	0	0
Roque^c	Lactating cows		Holstein-Friesian (729/759 kg)		A. armata – Bromoform (1.32 mg per g seaweed DM)
	17.3	8.3	1.84%	21	12	6.1	0.25	24.3	420	0.56
	24.9	11.5	0.92%	21	12	2.9	0.24	12.1	303	0.41
	27.9	14.4	0.00%	21	12	0	0	0	0	0.00
Roque^d	Feedlot steers		Hereford (352/583 kg)		A. taxiformis – Bromoform (7.8 mg per g seaweed DM)
Low Forage
High Asp	10.7	2.5	0.83%	63	7	9.9	0.15	64.4	689	1.47
Low Asp	10.8	3.8	0.45%	63	7	8.6	0.25	35.0	378	0.81
Control	11.5	12.4	0.00%	63	7	0	0	0	0	0.00
Med Forage
High Asp	10.0	3.9	0.83%	21	7	15.3	0.24	64.4	644	1.38
Low Asp	10.8	10.6	0.45%	21	7	8.6	0.25	35.0	378	0.81
Control	12.2	19.2	0.00%	21	7	0	0	0	0	0.00
High Forage
High Asp	8.4	10.6	0.83%	63	7	11.5	0.18	64.4	541	1.16
Low Asp	9.7	14.9	0.45%	63	7	7.2	0.21	35.0	340	0.73
Control	10.3	22.1	0.00%	63	7	0	0	0	0	0.00
Fennessy^e	Wether sheep		Romney-cross (42 kg)		A. armata – Bromoform (6.6 mg per g seaweed DM)
	0.89	2.4	0.61%	16	5	15.9	0.37	42.5	36	0.86
	0.88	4.8	0.41%	16	6	13.5	0.50	27.1	24	0.57
	0.96	14.5	0.19%	16	6	3.8	0.30	12.8	12	0.29
	0.87	18.3	0.00%	16	5	0	0	0.0		0.00
	Oil-based Asparagopsis system
Alvarez-Hess et al^f	Lactating cows (fed twice daily)		Holstein-Friesian (578 kg)		A. armata – Bromoform (480 mg/cow/day in canola oil ex steeped A. armata)
	30.1	24.6	NA	27	13
	28.4	13.8			11	11.4	0.67	16.9	480	0.83
	28.8	16.4			11	9.9	0.59	16.7

^a Stefenoni et al is not included as bromoform content is not presented; Muizelaar et al. is not included as there are no data on methane emissions; Li et al (2018) is not included as the bromoform content of the Asparagopsis is not defined.

^b https://doi.org/10.1016/j.jclepro.2020.120836; Methane – open-circuit chambers, CSIRO Lansdown Research Station; feed intake – physical.

^c https://doi.org/10.1016/j.jclepro.2019.06.193; Feed intake – Calan gates; Methane - GreenFeed, C-Lock, Inc., Rapid City, South Dakota, USA.

^d https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0247820; Methane – GreenFeed; Feed intake – Calan gates

^e Methane – open-circuit respiration chambers, AgResearch, Palmerston North, New Zealand; feed intake – physical.

^f https://www.sciencedirect.com/science/article/abs/pii/S0377840123000135?dgcid=author; Methane – modified SF6 (sulphur hexafluoride) tracer technique; Feed intake – Gallagher bin weight system.

 

The revised section is as follows:

 

As there were insufficient data to conduct a meta-analysis, we analysed the available data in a number of ways to ascertain the most appropriate form of expression. The mean effect of bromoform on methane reduction averaged –0.33 ± 0.15 (standard deviation) grams of methane per mg of bromoform consumed (n of 16 groups of animals). When the data are expressed as a dose response by regression (Figure 1), the equation (with the intercept constrained to zero) is:

Methane reduction (g methane per kg DMI) = −0.25*Bromoform intake (mg bromoform per kg DMI); n = 23; SE of the slope = ± 0.023; r² = 0.84; Residual SD = ± 3.19 (Equation 1).

Thus, the regression coefficient of −0.25 grams of methane per mg of bromoform intake is slightly lower than the mean reduction of −0.33.

Figure 1. The relationship between the reduction in methane emissions and the bromoform intake for five sets of data (the full data are in Table 1).

Given that the basal diet impacts the level of methane, the regression relationship between methane emissions (g methane per kg DMI) and bromoform intake is of interest. The regression relationship was:

Methane (g per kg DMI) = -0.19*Bromoform intake (mg bromoform per kg DMI) + 15.2; r² = 0.39; Residual SD = ± 4.99 (Equation 2).

A comparison of the variances for the two regression relationships indicates that the reduction in methane as a function of bromoform intake provides a better estimate of the efficacy of bromoform as the 59% reduction in variance from 24.85 (4.99²) in Equation 2 to 10.20 (3.19²) in Equation 1 was considered meaningful. In summary, while there must be a limit to the absolute scale of the methane response in that the baseline control value sets the upper limit on any response, a linear regression provides a reasonable description of the relationship in the range of data sets assessed.

Given the differences in concentrations of bromoform in the product used, the reduction in methane emissions is expressed on a dosage of bromoform basis (per mg bromoform), to enable more direct comparisons to be made between the different trials (as per Table 1).

• In a 90-day study, beef cattle showed a 98% reduction in methane when A. taxiformis (6.6 mg bromoform/gram), was included at 0.33% of feed dry matter (DM) (Kinley et al. 2020); the average reduction over three dose rates ranged from −0.50 to −0.18 g of methane per mg of bromoform, with an apparent dose response to the level of Asparagopsis in the diet, independent of the bromoform content.

• In a short 14-day study, lactating dairy cows showed a 20–43% reduction in methane when A. armata (1.3 mg bromoform/gram) was included at 0.92% or 1.84% of dry matter (DM), (Roque et al. 2019); the response was equivalent to −0.25 g reduction in methane per mg of bromoform; however, in this study, there were detrimental effects on feed intake.

• In a 147-day study, beef cattle showed a 33–80% reduction in methane when A. taxiformis (7.8 mg bromoform/gram), was included at 0.45% or 0.83% of feed DM on three different diets (Roque et al. 2021); the response was equivalent to −0.15 to −0.25 g reduction in methane per mg of bromoform.

• In a recent 16-day study with sheep fed A. armata (6.6 mg bromoform per gram of Asparagopsis dry matter fed once daily), Fennessy et al (in preparation) found an average reduction in methane equivalent to −0.40 g per mg of bromoform; the sheep were fed 13 to 43 mg bromoform per kg dry matter intake; there was no effect on feed intake.

• In a 27-day study in lactating cows with A. armata steeped in edible oil (ASP-Oil) to extract and stabilise bromoform, two formulations were tested – one with the Asparagopsis biomass included and one with biomass removed (Alvarez-Hess et al. 2022). They found reductions in methane equivalent to −0.63 g methane per mg bromoform.

Citation P. 12

On p.12 there is an error in a citation. The reference provided to support the statement “In nature, these bromo-compounds occur naturally in a diverse range of microalgae (Laturnus et al) has been replaced by (Shibazki et al. 2016, Sturges et al. 1992). The Laturnus et al. paper focuses on macroalgae not microalgae.

 

The revised section is as follows:

In nature, these bromo-compounds occur naturally in a diverse range of microalgae (Shibazki et al. 2016, Sturges et al. 1992).

 

The additional references are:

Shibazaki A, Ambiru K, Kurihara M, Tamegai H, Hashimoto S. 2016. Phytoplankton as a temperate marine source of brominated methanes. Marine Chemistry 181: 44–50. https://doi.org/10.1016/j.marchem.2016.03.004.

 

Stefenoni HA, Räisänen SE, Cueva SF, Wasson Sturges W, Cota G, Buckley P. 1992. Bromoform emission from Arctic ice algae. Nature 358: 660–662. https://doi.org/10.1038/358660a0.

Genetic toxicity and carcinogenicity P.17

New important genotoxicity data has come to light, which extends the information provided on p.17.

 

The revised section is as follows:

It is apparent that the supplier of chemical reagents, Sigma-Aldrich Merck has addressed the inconsistencies in historical testing dating back to the 1970s given new information now presented in their 2024 Safety Data Sheet for bromoform (Sigma-Aldrich 2024). They cite the results of six genetic toxicity tests from studies conducted to current OECD guidelines likely to be as part of in-house processes which include toxicology assessment of products listed in their catalogue. Unfortunately, these reports are not published but the safety data sheet information is informative: five out of six of these studies provided “negative” outcomes indicating a lack of genotoxicity.¹

 

In an early response to concern regarding drinking water safety, bromoform had been assessed and compared with other disinfection by-products for their mutagenic potency. The order of the mutagenic potency was 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone > bromoacetic acid > dibromoacetic acid > dichloroacetic acid > chloroacetic acid; tribromoacetic acid, trichloroacetic acid, bromoform, and chloroform CF were assessed as not mutagenic (Kargalioglu et al. 2002). These results align with the recently summarised results in the Sigma-Aldrich Safety Data Sheet on bromoform (Sigma-Aldrich 2024).

 

The added references are:

Kargalioglu, Y., McMillan, B., Minear, R. & Plewa, M. 2002 Analysis of the cytotoxicity and mutagenicity of drinking water disinfection by-products in Salmonella typhimurium. Teratogenesis, Carcinogenesis and Mutagenesis 22 (2), 113–128.

 

Sigma-Aldrich (2024) Safety Data Sheet Bromoform Version 6.7 Revision Date 07.03.2024 Print Date 24.03.2024 https://www.sigmaaldrich.com/NZ/en/sds/aldrich/241032?userType=undefined.

 

Reference

The report by Faust (1995) is no longer available on-line; therefore, the reference has been modified as below.

 

Faust RA. 1995. Toxicology Profiles. Formal Toxicity Summary for Bromoform. The Risk Assessment Information System. Chemical Hazard Evaluation Group, Biomedical and Environmental Information Analysis Section, Health Sciences Research Division, Oak Ridge, Tennessee (no longer available online)

Notes

1 Test Type: Ames test: S. typhimurium Metabolic activation: with and without metabolic activation Method: OECD Test Guideline 471 Result: positive.

Test Type: sister chromatid exchange assay test system: Chinese hamster ovary cells Metabolic activation: Metabolic activation Method: OECD Test Guideline 479 Result: negative.

Test Type: In vitro mammalian cell gene mutation test system: mouse lymphoma cells Metabolic activation: Metabolic activation Method: OECD Test Guideline 490 Result: negative.

Test Type: Chromosome aberration test in vitro test system: Chinese hamster ovary cells Metabolic activation: with and without metabolic activation Method: OECD Test Guideline 473 Result: negative.

Test Type: unscheduled DNA synthesis assay Species: Rat Application Route: Oral Method: OECD Test Guideline 486 Result: negative.

Test Type: In vivo micronucleus test Species: Mouse Application Route: Oral Method: OECD Test Guideline 474 Result: negative.